We subsequently tested the functional consequences

We subsequently tested the functional consequences of this fibrogenic plasticity in dystrophic muscle. The fluorescence-activated cell sorting (FACS)-isolated vegf inhibitor of endothelial and myogenic origins (YFP+) that had reduced (or lost) expression of CD31 or α7-INTEGRIN (YFP+CD31low/− or YFP+α7low/−) showed a severely impaired capacity to form angiotubes and myotubes in pro-angiogenic and pro-myogenic differentiation conditions, respectively (Figure 4G). Despite the significant fraction of both endothelial and myogenic cells undergoing fibrogenic plasticity in dystrophic muscle, surprisingly, each cell type only constituted about 2% of the bona fide active collagen-expressing cell population (i.e., active fibroblasts), based on intracellular collagen protein staining of the YFP+CD31low/− and YFP+α7low/− cell populations, respectively (Figure S5A). Similarly, using a ColI-GFP reporter mice (in which GFP expression is under the control of the Collagen I promoter), only a low percentage of CD31+ and α7-INTEGRIN+ cells were found within the fibroblastic (GFP+) cell population (Figure S5B). These findings strongly suggest that, unlike other fibrotic organs, such as kidney (LeBleu et al., 2013), these fibrogenic changes do not lead to full and ample transformation into collagen-producing cells. Instead, this fibrogenic plasticity mainly precludes efficient myogenesis and angiogenesis and it impairs tissue repair. We next investigated if endothelial and satellite cells also transit through intermediate mesenchymal progenitor states during the process of fibrogenesis in diseased muscle. To this end, we set up a FACS protocol based on the use of the cell surface mesenchymal progenitor marker PDGFRα (Chong et al., 2013; Pinho et al., 2013; Uezumi et al., 2010, 2014). A subpopulation of YFP+ cells that were low for CD31 or α7-INTEGRIN expression appeared positive for PDGFRα (YFP+PDGFRα+) in fibrotic muscles of dystrophic lineage-tracing mice (Figures 5A and 5B). The YFP+PDGFRα+CD31low/− and YFP+PDGFRα+α7low/− populations represented 14.6% and 16.3% of the fibrogenic YFP+CD31low/− and YFP+α7low/− cell fractions in adult mdx muscle, respectively (Figures 5A and 5B), and these percentages even increased in older mice (Figure S5C). As for the YFP+CD31low/− and YFP+ α7low/− cell fractions, the PDGFRα+-expressing cell subpopulations were incapable of forming myotubes or angiotubes under appropriate differentiation conditions (not shown). The qRT-PCR analysis confirmed induction of mesenchymal progenitor markers in the freshly isolated YFP+PDGFRα+CD31low/− and YFP+PDGFRα+α7low/− subpopulations from dystrophic muscle (Figure 5C). Thus, during the process of endothelial and satellite cell plasticity toward fibrogenesis, a fraction of cells shows mesenchymal progenitor traits. This was consistent with a subpopulation of YFP+ endothelial or myogenic cells also gaining PDGFRα+ expression in WT muscle subjected to CTX/TGFβ injury (Figure 5D). Consistent with the notion that TGFβ signaling is a driving cause for these plastic mesenchymal transitions, we could detect co-expression of activated SMAD2/3 in PDGFRα+/YFP+ cells in muscle of the distinct lineage-tracing mice (in various muscle degeneration/fibrosis paradigms) (Figure 5E; Figure S5D; data not shown) and in human DMD (see below). To finally prove that these subpopulations of YFP+ PDGFRα+ cells (from endothelial or myogenic origin) are indeed mesenchymal in nature, we tested their multipotency (the capacity to be coaxed to differentiate into distinct terminal fates: fat, bone, cartilage, or scar/fibrous) if exposed to adequate conditions. YFP+PDGFRα+ cells were FACS isolated from muscle of lineage-tracing dystrophic mice and subsequently cultured with osteogenic, adipogenic, or chondrogenic differentiation media or with TGFβ (for fibrogenic differentiation). In response to these treatments, sorted cells that had gained PDGFRα+ expression (YFP+PDGFRα+ cells), but not YFP+PDGFRα− cells, were positive for oil red staining (adipocyte), alkaline phosphatase staining (osteoblast), or collagen staining (fibroblast) (Figures 5F and 5G). In contrast, YFP+ cells, which were PDGFRα−CD31low/− or PDGFRα−α7low/−, did not show multipotency (not shown), indicative of a more differentiated fibrogenic state. These results suggest that the process of fibrogenic plasticity of endothelial and myogenic cells within dystrophic muscle, as with the plastic response to TGFβ in vitro (see Figure 1), involves multipotent progenitor cell intermediate states. Of note, in vivo interference with PDGFRα signaling with imatinib (a tyrosine kinase inhibitor), which has been shown to target PDGFRα-expressing mesenchymal progenitor cells (Ito et al., 2013; Uezumi et al., 2014), prevented the loss of cell identity in injured muscle in response to TGFβ (Figure S5E).