In the present study, we support other reports on the existence of adult fibrogenic EMT of the EMCs lining the epicardium 
and propose a model of how fibrogenic EMT of EMCs is controlled at the level of miRNAs (). We implicate TGF-β as a far more potent EMT-inducer in EMCs than TNF-α and especially IL-1β, and detected substantial differentiation into myofibroblastic cells for TGF-β incubation, as measured by various markers, including development of stress fibers of α-SMA (). Despite this differential potency in inducing EMT, miR-21 was the most up-regulated miRNA induced by all three stimuli.
Notably, IL-1β and TNF-α induced miR-21 at equal levels (), but TNF-α was far more potent in inducing EMT, which is likely an outcome of differential induction of the molecular pool unrelated to miR-21 effects. As such, the more diverse TNF-α signaling, including caspase 8-mediated death signaling 
, might promote EMT more compared to the classic NF-kB/MAPK pathways of IL-1β 
, or that IL-1β also induces factors that act to hamper EMT, while TNF-α may not.
Importantly, miR-21 manipulations showed substantial impact on EMC phenotype, since overexpression of miR-21 in EMCs markedly promoted biomarkers of fibrogenic EMT, while fibrogenic EMT was substantially inhibited when a miR-21 antagonist was present during EMT (). We observed more impact from the miR-21 antagonist than the miR-21 mimic. This is likely because anti-miR-21 transfection was performed 20h after EMCs had undergone TGF-β treatment and readouts assessed after another 24h of incubation in TGF-β, while Pre-miR-21 transfection was performed on resting EMCs and the phenotype was evaluated after 48h.
Our results indentify miR-21 as a key regulator of fibrogenic EMT of EMCs, which is in parallel to an increasing number of reports documenting high significance of miR-21 in cardiac diseases. As reported by several independent groups, analyses on both patient samples 
along with animal injury models 
, miR-21 is implicated in cardiac hypertrophy, proliferative vascular disease and ischemic heart disease. We furthermore show that miR-21 is up-regulated in a time-dependent manner during TAC () and LAD (), and not only in later strict fibrosis-associated phases, but that miR-21 up-regulation is already substantial at very early time-points dominated by inflammatory phases, characterized by e.g. high levels of pro-inflammatory molecules such IL-1β 
and TNF-α 
. However, recent contradictory results on the involvement of miR-21 in cardiac disease imply that more studies are needed to elucidate miR-21′s role in the heart 
Nevertheless, miR-21 has been associated with cell survival and hypertrophy in cardiomyocytes 
, whereas in vascular smooth muscle cells and fibroblasts, miR-21 promotes cellular activity in the direction of remodeling, which can be reduced or even reversed by miR-21 antagonists 
, supporting our results. Additionally, we show that the miR-21-mediated PDCD4- and SPRY1-targeting promotes a number of mesenchymal, fibrogenic markers in EMCs. We therefore propose that miR-21 mediates cardiac remodeling also through EMT of EMCs, partly through a PDCD4- and SPRY1-dependent mechanism (). As such, miR-21 may skew the EMC progenitor potential towards remodeling, rather than regeneration.
At the molecular level, several miR-21 targets have been experimentally validated in several biological systems. In cardiac disease, functional assessment of miR-21 implicates PDCD4 
, SPRY1 
or SPRY2 
, Phosphatase and tensin homolog 
, and Fas ligand 
as the major targets of miR-21. Our data solidly support the involvement of PDCD4 and SPRY1 in miR-21-regulated fibrogenic processes in the heart. However, the phenotypic measurements also suggest that other targets of miR-21 are involved in cardiac fibrogenic EMT, since e.g. α-SMA was regulated through a miR-21-dependent mechanism (), which could not be proven to occur through PDCD4 or SPRY1 ( and ). This supports the notion that a single miRNA may regulate several target mRNAs 
and furthermore supports that miR-21 is a pleiotropic acting miRNA within biological systems such as cancers and cardiovascular diseases 
. In this study, we focused on miR-21 targets relating downstream to the MAPK/NF-κB pathways as all three tested EMT inducers can signal through those, but in relation to TGF-β-induced EMT it would be relevant to further explore miR-21 targets relating the TGF-β pathway to account for the missing functional effect from miR-21 at the level of downstream targets (). However, Smad7, which have been shown to be a miR-21 target in pulmonary fibrosis 
, does not appear to be implicated in our culture system, since we registered no significant regulation of Smad7 expression during EMT (Fig. S9
Our miRNA array data show many differentially regulated miRNAs besides miR-21 ( and Table S2
). However, the effects from miR-21 account for up to 50% or even more on the investigated fibrogenic EMT biomarkers (), substantiating that miR-21 is not only the most regulated miRNA, but also a functionally outmost important miRNA during cardiac fibrogenic EMT. Similar to our results, two reports on TGF-β-induced EMT in human keratinocytes 
and a colon carcinoma cell line 
both identified miR-21 as the major up-regulated miRNA during EMT, and a recent study on TGF-β-induced cardiac EndMT showed that miR-21 promoted EndMT through targeting of the PTEN/Akt pathway 
. In addition, miR-21 targeting of PDCD4 and RECK has been identified as a driver of branching morphogenesis of the epithelium in the submandibular gland 
. However, other in vitro
studies of TGF-β-induced EMT have identified miR-155, by RhoA targeting 
, and especially the miR-200 family in tumor metastasis, by targeting E-cadherin transcriptional repressors ZEB1/ZEB2 
, as EMT-promoting miRNAs. Owing to the different nature of the epithelial cell lines used in those studies compared to the mesothelial cardiac origin of the EMCs in our study, the miRNA fingerprint along with the differential miRNA expression during EMT is expected to differ substantially. Furthermore, since rat-derived EMCs were used, and miR-155 is not annotated in the rat (miRBase 10.0–15.0), we could not assess miR-155 in our analyses.
In summary, miR-21 promotes robust fibrogenic EMT of EMCs, in part by directly targeting PDCD4 and SPRY1. Due to the multipotency of EMCs, our results implicate miR-21 as a relevant target to reduce the differentiation of EMCs into fibroblast-like cells, which may favor commitment into the vasculogenic/angiogenic or even the myogenic lineage.