The comparative transcriptome analysis described here demonstrates a unique molecular signature for induced regeneration-competent (iRC) fibroblasts compared with control fibroblasts. Consistent with the notion that these two cell types are distinctly different, we have used both cell types in in vivo regeneration experiments and demonstrated that the induced regeneration-competent fibroblasts participate in regenerative response of skeletal muscle (concomitant with decreased scar formation), contribute to the pool of newly established satellite cells (PAX7+ cells) in a mouse injury model, as well as form mature myotubes .
Identification of significantly differentially expressed genes and subsequent Gene Ontology analysis determined that a large number of genes important for the outcome of wound healing such as extracellular matrix genes, adhesion molecules, matrix remodeling genes, and genes involved in inflammation were regulated by FGF2 (Figure 3).
During dermal wound healing, fibroblasts are responsible for ECM production  and, here, we show that FGF2 treatment affects a number of genes involved in production and remodeling of ECM. FGF2 caused downregulation of a number of collagens such as collagen IV, collagen XI, collagen V, and collagen I, as well as caused upregulation of collagen XXI and collagen XIV (Table 1). qRT-PCR analysis confirmed downregulation of COL1A1, COL4A1, COL4A2, COL4A4, COL8A1, and COL11A1 (Figure 4B). FGF2 was previously shown to downregulate expression of interstitial collagen I and III . Collagen I is a major component of ECM in skin, and during wound healing is the main scar forming collagen. Collagen IV is a major constituent of basement membrane (other components include laminin, nidogen, and heparan sulfate proteoglycan perlecan) and is a predominant type of collagen found in skeletal muscle. Other ECM genes affected by FGF2 treatment included laminins and fibronectins (Table 1). Most profoundly affected by FGF2 treatment were laminin gamma 1 (LAMC1) and laminin alpha 5 (LAMA5). qRT-PCR confirmed increased expression levels of these two laminins (Figure 4B). Fibronectin 1 was downregulated by FGF2 treatment (Figure 4B). FGF2 treatment of human fibroblasts modulates production of the ECM. The ECM composition of the FGF2-treated fibroblasts favors the pro-regenerative outcome in the wound site directly by affecting the balance between scar formation and tissue regeneration and potentially thorough changes in cell attachment to ECM, cell migration, and cell proliferation.
Cell attachment to the ECM is regulated through integrins, heterodimers that recognize specific substrates. Adhesion and migration on collagen substrate is performed through α1β1 and α2β1 and formation of collagen type I and type III network is dependent on fibronectin and α2β1 [25–27]. We show FGF2-induced upregulation of β1 and α2 (Figure 4B). Integrins α5β1, αVβ3, and α4β1 pairs are utilized to bind fibronectin matrix [28, 29], αVβ5 is used to adhere to vitronectin, and α6β1, α2β1, α3β1 to adhere to laminin and entactin [10, 12, 30]. FGF2 treatment downregulated ITGB2 and upregulated ITGB3 and ITGA10 (Figure 4B). Integrins connect ECM to actin cytoskeleton via focal adhesions rich in talin, which is recruited to F-actin, and binds integrin pairs, which in turn leads to transmission of F-actin movements to ECM . Change in the composition of integrins, as well as in the components of focal adhesions leads to change in migration, as well as preferential binding to specific substrate, production of which is regulated by FGF2 treatment, and may benefit a pro-regenerative response.
During wound healing, fibroblasts acquire a highly migratory phenotype. The process is driven by actin polymerization and resulting microfilaments of the cell’s leading edge link to ECM via integrins. Actomyosin contraction then allows for the disassembly of adhesions in the rear and movement forward . Thus, movement of the fibroblasts in the wound site is regulated not only by the ECM and adhesion molecules, but also by the actin cytoskeleton. Actin cytoskeleton is also involved in fibroblast contractile phenotype. During dermis healing, fibroblasts generate stress fibers (weakly contractile actin bundles) to enable contraction . Fibroblasts’ shape is regulated by the environment and cell-matrix adhesion determines the cell shape, such as strong cell-ECM adhesion promotes spindle-shaped fibroblast . In vitro fibroblasts were shown to have different morphology depending on the substrate they are grown on; in 3D cultures resembling an in vivo environment, fibroblasts display elongated shape, well-developed actin cortex, and filopodia at the leading edge . Alpha actin ACTC1, which is a constituent of the contractile apparatus, was downregulated in human dermal fibroblasts treated with FGF2 (Table 2; Figure 5B). Gamma actin ACTG2, which is involved in cellular motility and adhesion, was 64-fold downregulated (Table 2), though qRT-PCR did not confirm its expression. By regulating cytoskeleton gene expression, FGF2 potentially promotes cell migration in the wound site, and reduces contraction that leads to the favorable pro-regenerative outcome.
Previously, it was shown that administration of FGF2 alone into a dermal wound shows reduced scar formation , which can be attributed to upregulation of matrix metalloproteinase MMP1 . Our data shows strong upregulation of MMP1 (Figure 4B), the metalloproteinase responsible for cleaving collagen type I, II, and III . FGF2 signaling was shown to activate the MMP1 promoter . MMP1 was able to improve the skeletal muscle regeneration process by reducing scar tissue formation [36–38] and by promoting migration of myoblasts involved in regeneration of skeletal muscle [39, 40]. Interestingly, integrin α2β1 was shown to increase MMP1 expression [41, 42]. By transplanting FGF2 treated human dermal fibroblasts, continuous increase in production of MMP1 among other factors, may be allowed, indicating that MMP1 is present not only at the time of the resolution phase of wound healing leading to decreased collagen production, but also at earlier stages of wound healing, for example during the inflammation stage. Other MMP molecules, such as stromelysins MMP3, MMP10, MMP11, were upregulated as well (Table 1 and Figure 4). MMP3 was previously shown to be responsible for contraction of fibroblasts during wound healing  and was regulated by FGF2 in a mouse model . MMPs, mostly MMP2, 3, 9 and 10, are highly upregulated during amphibian limb regeneration [45–47]. All of these observations point toward a favorable role of MMPs in the regeneration process. Thus, FGF2-stimulated change in transcriptional profile of various MMPs is an important factor contributing to the regeneration-competence of fibroblasts.
FGF2 treatment also led to a favorable ratio between MMPs and tissue inhibitors of metalloproteinases (TIMPs), as an imbalance between MMPs and TIMPs has been shown to increase scar formation. FGF2 upregulated TIMP4 and downregulated TIMP3 expression (Figure 4B). ADAM and ADAMTS proteinases that were shown to be differentially regulated by FGF2 (Table 1) are regulators of ECM and adhesion molecules and affect cell motility, adhesion, and signaling during wound healing processes. ADAMTS1 and ADAMTS5 were downregulated by FGF2 treatment (Figure 4B). ADAM transmembrane proteinases are involved in cleaving and activating various cell surface molecules, whereas ADAMTS are secreted proteinases that can bind ECM. ADMATS8 that was upregulated by FGF2 treatment (Figure 4B) has anti-angiogenic properties .
The ratio of TGFB1/TGFB3 is a factor that predicts scar formation, the decrease in this ratio being indicative of reduced scar formation . Fetal wounds that are known to heal without scar formation exhibit decreased TGFB1 levels . Administration of TGFB3 has also been shown to reduce scar formation . TGFB pathway was previously shown to be induced by FGF2 treatment in mouse embryonic fibroblasts (MEFs) . We observed no change in the levels of TGFB1 due to FGF2 treatment by microarray analysis whereas qRT-PCR showed downregulation of TGFB1 levels (Figure 5B). We observed upregulation of TGFBR3 due to FGF2 treatment by the array, but qRT-PCR showed no change in expression levels (Figure 5B). qRT-PCR confirmed downregulation of TGFBR1 (Figure 5B). These observations may be due to differences between mouse embryonic fibroblasts and adult human dermal fibroblasts, indicating that FGF2 response in these cells may be unique.
Decreasing inflammation has been shown to decrease scar formation. For example, when wounds of skin and oral mucosa were compared, there was less inflammation and scarring in oral mucosa . Non-scar wound healing in fetal wounds is also characterized by absence of inflammation [52–56]. Inflammatory events are integrated by chemokines. Chemokines are chemotactic cytokines that regulate migration of cells during inflammatory process. ELR(+) CXC chemokines are neutrophil attractants and activators. CXCL6 or granulocyte chemotactic protein-2 (GCP-2) is a ELR(+) CXC chemokine. FGF2 treatment led to increase in CXCL6 chemokine expression (Figure 5B). CXCL5, a chemokine that attracts and activates neutrophils, amplifies inflammatory cascade, and stimulates local production of cytokines was shown to be upregulated by FGF2 treatment (Figure 5B). Interestingly, when CXCL5 is cleaved by MMP1, 2, 8, 9, and 13, increased inflammation is observed and cell recruitment to the wound site is activated . CCL2 (monocyte chemoattractant protein-1, MCP-1), which is involved in inflammatory cell recruitment, can be induced through focal adhesion kinase (FAK) leading to inflammation and scar production in a cutaneous injury, and CCL2 knock-out mice showed decreased scarring . Here, we observed downregulation of CCL2 due to FGF2 treatment (Figure 5B). In agreement with previous publications, implantation of FGF2 treated fibroblasts, which show CCL2 downregulation, into a mouse wound sight leads to reduced scar formation . We also show in this transcriptome analysis that IL6/STAT3 signaling pathway is regulated by FGF2 (Figure 5B). Interleukin 6 (IL6) is a pleiotropic cytokine that is produced by a variety of cells such as epidermal cells, endothelial cells, and fibroblasts . IL6 is known to increase production of collagen , thus the decrease in collagen synthesis that we observe in skeletal muscle injury, can be partially explained by decrease in IL6. CCL2 was shown to induce IL6 secretion in human lung fibroblasts, and has a role in regulating fibrosis  and was shown previously to be regulated by FGF2 . Scarless, fetal wounds are characterized by diminished expression of pro-inflammatory IL6 and IL8 [52, 53]. Here, we show that FGF2 treatment significantly reduces IL6 levels (Figure 5B), whereas levels of IL8 are upregulated with FGF2 treatment (Table 3). FGF2-induced decrease in IL6 level could be contributing to pro-regenerative phenotype of adult human fibroblasts. Signal transducer and activator of transcription (STAT3) conveys signals from IL6. Loss of IL6 was shown to result in deficiency of proliferation and migration of myoblasts [63–65]. IL6/STAT3 was shown recently to be involved in excessive ECM production and increased cellular proliferation in hypertrophic scars compared to normal human fibroblasts .