**Platelet-Rich Plasma Modulates Gap Junction Functionality and Connexin 43 and 26 Expression During TGF-**β**1–Induced Fibroblast to Myofibroblast Transition: Clues for Counteracting Fibrosis**

**Roberta Squecco 1,**†**, Flaminia Chellini 2,**†**, Eglantina Idrizaj 1, Alessia Tani 2,**†**, Rachele Garella 1, Sofia Pancani 2, Paola Pavan 3, Franco Bambi 3, Sandra Zecchi-Orlandini <sup>2</sup> and Chiara Sassoli 2,\*,**†


Received: 7 April 2020; Accepted: 8 May 2020; Published: 12 May 2020

**Abstract:** Skeletal muscle repair/regeneration may benefit by Platelet-Rich Plasma (PRP) treatment owing to PRP pro-myogenic and anti-fibrotic effects. However, PRP anti-fibrotic action remains controversial. Here, we extended our previous researches on the inhibitory effects of PRP on in vitro transforming growth factor (TGF)-β1-induced differentiation of fibroblasts into myofibroblasts, the effector cells of fibrosis, focusing on gap junction (GJ) intercellular communication. The myofibroblastic phenotype was evaluated by cell shape analysis, confocal fluorescence microscopy and Western blotting analyses of α-smooth muscle actin and type-1 collagen expression, and electrophysiological recordings of resting membrane potential, resistance, and capacitance. PRP negatively regulated myofibroblast differentiation by modifying all the assessed parameters. Notably, myofibroblast pairs showed an increase of voltage-dependent GJ functionality paralleled by connexin (Cx) 43 expression increase. TGF-β1-treated cells, when exposed to a GJ blocker, or silenced for Cx43 expression, failed to differentiate towards myofibroblasts. Although a minority, myofibroblast pairs also showed not-voltage-dependent GJ currents and coherently Cx26 expression. PRP abolished the TGF-β1-induced voltage-dependent GJ current appearance while preventing Cx43 increase and promoting Cx26 expression. This study adds insights into molecular and functional mechanisms regulating fibroblast-myofibroblast transition and supports the anti-fibrotic potential of PRP, demonstrating the ability of this product to hamper myofibroblast generation targeting GJs.

**Keywords:** α-smooth muscle actin; confocal microscopy; connexin 43; connexin 26; fibrosis; gap junctions; myofibroblasts; Platelet-Rich Plasma; skeletal muscle; transforming growth factor (TGF)-β1

#### **1. Introduction**

Adult skeletal muscle can efficiently repair/regenerate after focal damages [1]. Several studies showed that many different cell types endowed with inducible myogenic potential, residing within the muscle tissue or recruited via the blood, might contribute to the formation of nascent contractile myofibers [2–6]. Nevertheless, muscle resident satellite cells are widely recognized as the main

players in the repair/regenerative processes [7,8]. After focal injuries, satellite cells undergo activation to essentially recapitulate the steps of embryonic and fetal myogenesis forming new myofibers or fusing with injured myofibers to repair the damage [1,9]. To accomplish their task, satellite cells (but even the myogenic non-satellite cells), require the establishment of a suitable and conductive surrounding microenvironment. This essentially includes pro-myogenic factors, biochemical and physical pro-myogenic signals, juxtacrine, and paracrine interaction with different interstitial nursing cells and a spatially and temporally limited reparative fibrotic response [1,10–15].

#### *1.1. Fibrotic Response in Skeletal Muscle*

The activation of fibrogenic pathways represents an adaptive physiological response of tissues, including skeletal muscle, to damage. A crucial process in such a fibrotic response is represented by the differentiation of fibroblasts resident in the extracellular matrix (ECM) towards myofibroblasts [16]. This is essentially promoted by the combined action of pro-fibrogenic agents, such as transforming growth factor (TGF)-β1, mainly released by infiltrating inflammatory cells (particularly macrophages) at the site of the injury and by the fibroblasts/myofibroblasts itself, and mechanical stimuli coming from the damaged microenvironment [16,17]. Myofibroblasts are characterized by a prominent rough endoplasmic reticulum, typical of collagen-synthetically active fibroblasts, by the de novo expression of α-smooth muscle actin (α-sma) within well-assembled stress fibers that confers contractile properties to the cells. Stress fibers are anchored to fibronexus, a specialized focal adhesion complex on the myofibroblast surface, to link intracellular actin filaments with extracellular fibronectin fibrils. Through fibronexus, the force generated by stress fibers can be transmitted to the surrounding ECM, and vice-versa the ECM mechanical signals can be transduced via this mechano-transduction system into intracellular signals [18]. Moreover, even if myofibroblasts are not regarded as electrically excitable cells, they show peculiar biophysical properties and trans-membrane ion currents typical of smooth muscle cells. In this regard, it has been reported that human atrial myofibroblasts can express a Na<sup>+</sup> current (INa) and biophysical properties that could give rise to regenerative action potentials [19,20]. In addition, myofibroblasts typically show the inward-rectifier K<sup>+</sup> current (Ikir), which especially increases under TGF-β1-treatment [21–24]. In physiological conditions, the permanence and function of myofibroblasts after muscle damage are temporally and spatially limited. Indeed, they are responsible for the deposition of ECM components to form a transient contractile scar essentially required to rapidly restore tissue integrity and preserve muscle function, to support activated SCs and the nascent myofibers mechanically. Once the tissue regeneration has taken place, the scar will be degraded thanks to the balanced and finely tuned activity of proteolytic enzymes selectively digesting individual components of ECM, namely matrix metalloproteinases (MMPs), and of their specific tissue inhibitors (TIMPs), that are mainly secreted by different cells including, among others, fibroblasts and inflammatory cells [25]. Myofibroblasts progressively disappear, undergoing apoptosis and/or senescence or reverting to a quiescent state [26,27]. By contrast, the persistence of myofibroblasts in an activated state has been associated with an aberrant maladaptive reparative response to chronic or extended damage leading to the formation of a permanent scar replacing the normal functional tissue and hampering the endogenous cell mediated-mechanism of muscle regeneration [10,16,17,28,29]. Therefore, therapies aimed to limit myofibroblast generation and functionality may result strategical and effective for preventing tissue fibrosis development and thus promoting the regeneration of damaged muscles.

#### *1.2. PRP as an Anti-fibrotic Agent*

In this regard, Platelet-Rich Plasma (PRP)—defined as a plasma fraction with a concentration of platelets above baseline levels and representing a source of numerous biologically active molecules—may offer promising perspectives [30]. Indeed, many in vitro and in vivo studies have demonstrated the anti-fibrotic potential of this blood product in different tissues [31–38], including skeletal muscle [30,39–46], and have indicated the fibroblast-myofibroblast transition as the cell process target of its action [31–33,36,38,44,47,48]. Furthermore, the positive contribution of PRP to skeletal muscle regeneration has been demonstrated either in vivo or in vitro, thanks to its capability to promote the myogenic program [30]. Nevertheless, the anti-fibrotic potential of PRP needs to be investigated more in-depth, and the molecular targets of the action of this plasma product need to be clearly identified.
