Platelet rich plasma prevents voltage-dependent gap junction currents and Connexin 43 expression in TGFβ1- induced myofibroblasts in vitro through Vascular endothelial growth factor (VEGF)-A Mediated Signaling

Although increasing evidence supports the antifibrotic potential of Platelet rich plasma (PRP), currently there is not a clear consensus and the cellular and molecular mechanisms, underpinning PRP action, need to be clearly identified. In this context, we recently demonstrated the capability of PRP to prevent the in vitro differentiation of fibroblasts towards myofibroblasts, the key cell effectors of fibrosis induced by the pro-fibrotic agent transforming growth factor (TGF)-β1. Moreover, PRP reduced the expression of α-smooth muscle actin (sma), a well-known myofibroblastic marker, engaging the vascular endothelial growth factor (VEGF)-A/ VEGF Receptor-1-mediated signaling. In addition, we demonstrat-ed the capability of PRP to abolish the TGF-β1-induced voltage-dependent gap junction (GJ) current appearance while preventing the expression of connexin (Cx) 43, the typical Cx isoform forming voltage-dependent connexons. Here we extended our research assessing the involvement of VEGF-A/ VEGF receptor signaling in mediating the in vitro PRP-induced response of fibroblasts cultured in the presence of TGF-β1 in terms of GJ currents and Cx43 expression, by com-bining morphological and electrophysiological analyses. First, we confirmed the ability of PRP to prevent the acquisition of myofibroblastic phenotype by TGF-β1-treated fibroblasts as judged by cell shape and surface area analyses, confocal immunofluorescence analysis of α-sma and vinculin expression and electrophys-iological recordings by whole‐cell patch‐clamp of the resting membrane potential. Furthermore, we found that VEGF-A neutralization by blocking antibodies or the VEGFR inhibition by KRN633 in TGF-β1-treated fibroblasts prevented the PRP-induced down-regulation on the GJ currents and Cx43 expression. The role of VEGF-A signaling in preventing voltage dependent GJ currents occurrence and CX43 expression upregulation was confirmed by the treatment of fibroblasts with soluble VEGF-A. This study contributes to provide new insights into the molecular mechanisms underpinning PRP action on counteracting fibroblast to myofi-broblast differentiation, recognizing GJs and Cx43 crucial targets of the VEGF-A mediated pathway.