*4.4. Factors Released by PRP Possibly Modulating Cx Expression and GJ Functionality During Myofibroblast Generation*

Finally, it is worth mentioning that the factors released by PRP possibly modulating Cx expression and GJ functionality during myo-differentiation process remain to be identified as well as their potential modes of action. We have previously demonstrated that PRP contains vascular endothelial growth factor (VEGF)-A and that, through this factor linking its receptor-1 (VEGFR-1 or Flt-1), PRP antagonizes TGF-β1/Smad3, thus preventing fibroblast myo-differentiation [30,48]. Taking into consideration that VEGF may modulate the expression of Cx43 and/or GJ functionality in different cell types [99–101] we may speculate that GJs/Cx43 might represent a downstream target of VEGF-A/VEGFR-1 mediated signaling in our experimental cell model. Studies are ongoing in our lab to assess this hypothesis. Moreover, it is known that GJ assembly and disassembly are events highly regulated by a sequence of protein kinase activation and phosphorylation events. This kind of regulation, as extensively reported for Cx43, has a net effect of reducing GJ communication [102–104]. Therefore, we may also postulate that VEGF-A or other factors released by PRP may reduce GJ/Cx43 functionality affecting such phosphorylation events. In such a view it has been reported that insulin-like growth factor (IGF)-1 is able to decrease gap junctional communication by inducing activation of PKCγ, enhancing the interaction between PKCγ and Cx43 and the phosphorylation of Cx43 by PKCγ [105] in epithelial cells. Since IGF-1 has been reported to be contained in PRP [106] and has been supposed as a potential modulator of fibrogenic events and pathways [14,107,108], it is tempting to speculate that similar interactions may also occur in our cell system. Therefore, the capability of PRP to counteract fibroblast

differentiation towards myofibroblasts and its ability to modulate GJIC could also involve the IGF-1 signaling pathway. A full characterization of the releasing profile of likely cross-talking factors present in PRP are required to understand the molecular mechanisms underpinning the action of this plasma product.

#### **5. Conclusions**

In conclusion, the results of the present in vitro study provide the first experimental evidence that upregulation of Cx43 and the parallel increase of voltage-dependent GJ functionality are important mechanisms by which TGF-β1 endorses fibroblast differentiation towards myofibroblast, and that PRP treatment hampers this effect.

The main limitations of this study rely on the in vitro experimentation on the NIH/3T3 cell line. Obviously, the in vitro experimentation eliminates many paracrine/juxtacrine mechanisms, possibly regulating in situ intercellular interactions and cell functionality as well as the mechanical forces exerted by the surrounding microenvironment, including ECM stiffness, affecting cell behavior [16,18,109]. NIH/3T3 cells represent a widely used, reliable model to study fibroblast biology. We previously demonstrated that these cells show similar behavior, in terms of differentiation marker expression and electrophysiological parameters, to primary fibroblasts, such as human dermal fibroblasts, skeletal, and cardiac fibroblasts [23,48,55,56]. Nevertheless, the growing evidence on functional heterogeneity and the origin-linked response of fibroblasts to stimuli must be taken into account [110–112]. Therefore, we acknowledge that a different experimental set using primary cultures of skeletal muscle-derived fibroblasts could have offered in vitro findings possibly more closely related to in vivo conditions of skeletal muscle disease and fibrosis.

Another limitation is represented by the lack of a full characterization of the growth factors released by PRP. This should be relevant to understand better PRP mechanisms of action in the modulation of fibroblast-myofibroblast transition and to achieve a therapeutic translation of this approach. Furthermore, standardization of PRP preparation techniques as well as application protocols would allow performing meaningful comparative analyses.

However, despite these aspects, this research contributes to add further insights into molecular and functional mechanisms regulating fibroblast-myofibroblast transition. It likewise supports the anti-fibrotic action of PRP by means of its ability to hamper myofibroblast generation, targeting GJs, thus providing cues to novel therapeutic targets.

**Author Contributions:** Conceptualization, R.S., F.C., E.I., A.T., and C.S.; formal analysis, R.S., F.C., E.I., A.T., and C.S.; investigation, R.S., F.C., E.I., A.T., R.G., S.P., and C.S.; resources, R.S., F.C., P.P., F.B., S.Z.-O., and C.S.; data curation, R.S., F.C., E.I., A.T., and C.S.; writing—original draft preparation, R.S., and C.S.; writing—review and editing, R.S., F.C., E.I., A.T., S.Z.-O., and C.S.; visualization, R.S., F.C., E.I., A.T., and C.S.; funding acquisition, R.S., F.C., S.Z.-O., and C.S. All authors have read and agreed to the published version of the manuscript."

**Funding:** This research was supported by the annual financing fund by MIUR (Ministry of Education, University and Research, Italy)—University of Florence to R.S., F.C., S.Z.-O., and C.S. and by FFABR-MIUR 2017 (Financing Fund for Basic Research Activities) granted to R.S. and C.S.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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