Endogenous Epicardial Regenerative Response Figure 4. Proliferation of epicardial and subepicardial cells upon myocardial ischemia. In control hearts BrdU was not detected in the epicardium covering the atrioventricular junction (A), left ventricle (B) and atrium (C). As a positive control, BrdU incorporation was confirmed in the crypts of the gut in each mouse (D). At two days post-MI BrdU was detected in Wt1-positive epicardial cells (arrows) and some Wt1-negative cells throughout the myocardium of the apex (E). The thin dotted line highlights the border between the epicardium and underlaying myocardium. At three days post-MI BrdU-positive cells are found within in the epicardium covering the infarcted area (F), atria (G), and in mesenchymal cells in the border zone of the infarct (H). At one week post-MI BrdU was detected in cells located in the subepicardial space (double arrow) covering the infarcted area (I, J). At two weeks post-MI, BrdU was detected in a few individual subepicardial cells of the atria (arrow) (K) and of the border zone of the infarct (L). At one and three months post-MI hardly any BrdU incorporation was detected in the heart (M–P). Abbreviations: LA; left atrium, LV: left ventricle, RV: right ventricle, AVS; atrioventricular junction, Epi:epicardium, Subepi: subepicardial mesenchyme. The bar is 100 mm. doi:10.1371/journal.pone.0044692.g004 both the epicardial and mesenchymal cells. In areas remote of the infarct and in controls expression of Snai1 was not observed. At two weeks post-MI, the number of cells expressing these markers had decreased and at three months post-MI only a few scattered mesenchymal cells were found in the border zone. Because Snai1 is closely related to Snai2, we prepared primers to amplify the two genes separately. The qPCR analysis showed that Snai1 mRNA is not detectable in control hearts, was induced at 1 day post MI, and starts to decrease after twee weeks post-MI, to return to almost undetectable levels at three months post-MI (Figure 5C). Its relative Snai2 was detected in control hearts, and follows a similar temporal expression profile as Snai1. To exclude that the primers were not sufficiently specific, the PCR products were cloned and sequenced. Taken together these findings further underscore our interpretation that at least a subset, if not all, mesenchymal cells found between the epicardial layer and the infarcted myocardium are formed by EMT from the overlaying epicardium. Another group of proteins regulating EMT are matricellular proteins [40,41]. These proteins function by their ability to interact with multiple cell surface receptors, as well as by binding directly to structural or scaffold proteins. The expression of this family of proteins is most prominent during development but has also been shown to play an important role in the response to injury [40]. We investigated the expression of two members of this PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e44692