Platelet-Rich Plasma and Bone Marrow-Derived Mesenchymal Stromal Cells Prevent TGF-β1-Induced Myofibroblast Generation but Are Not Synergistic when Combined: Morphological in vitro Analysis

Abstract

The persistence of activated myofibroblasts is a hallmark of fibrosis of many organs. Thus, the modulation of the generation/functionality of these cells may represent a strategical anti-fibrotic therapeutic option. Bone marrow-derived mesenchymal stromal cell (MSC)-based therapy has shown promising clues, but some criticisms still limit the clinical use of these cells, including the need to avoid xenogeneic compound contamination for ex vivo cell amplification and the identification of appropriate growth factors acting as a pre-conditioning agent and/or cell delivery vehicle during transplantation, thus enabling the improvement of cell survival in the host tissue microenvironment. Many studies have demonstrated the ability of platelet-rich plasma (PRP), a source of many biologically active molecules, to positively influence MSC proliferation, survival, and functionality, as well as its anti-fibrotic potential. Here we investigated the effects of PRP, murine and human bone marrow-derived MSCs, and of the combined treatment PRP/MSCs on in vitro differentiation of murine NIH/3T3 and human HDFα fibroblasts to myofibroblasts induced by transforming growth factor (TGF)-β1, a well-known pro-fibrotic agent. The myofibroblastic phenotype was evaluated morphologically (cell shape and actin cytoskeleton assembly) and immunocytochemically (vinculin-rich focal adhesion clustering, α-smooth muscle actin and type-1 collagen expression). We found that PRP and MSCs, both as single treatments and in combination, were able to prevent the TGF-β1-induced fibroblast-myofibroblast transition. Unexpectedly, the combination PRP/MSCs had no synergistic effects. In conclusion, within the limitations related to an in vitro experimentation, our study may contribute to providing an experimental background for supporting the anti-fibrotic potential of the combination PRP/MSCs which, once translated “from bench to bedside,” could potentially offer advantages over the single treatments.

Platelet-Rich Plasma Modulates Gap Junction Functionality and Connexin 43 and 26 Expression During TGF-β1–Induced Fibroblast to Myofibroblast Transition: Clues for Counteracting Fibrosis

“…In such a context, several in vitro and in vivo studies provided evidence supporting the advantage of the use of PRP for muscle regenerative purpose [30,66]. In this line, we have recently demonstrated the capability of PRP to either stimulate proliferation and differentiation of myogenic progenitors, including satellite cells [58], or prevent the TGF-β1 induced differentiation of fibroblasts towards myofibroblasts [48,57].…”

“…In particular, we focused the attention on the GJIC. The experimental model to evaluate fibroblast to myofibroblast transition has been previously validated [23,24,48,[55][56][57] and consists in the culture of the cells in low serum conditions in the presence of TGF-β1. The treatment with PRP was also conducted as previously reported [48,57,58].…”

“…For the present experiments, thawed ready-to-use activated PRP aliquots classified as not suitable for transfusion-infusion purposes previously prepared and stored at −80 • C were used [48]. Briefly, PRP was collected from the blood of healthy adult donors subjected to plasma-platelet apheresis (Haemonetics MCS®, Haemonetics, Milan, Italy) as previously reported in detail [57]. The final platelet concentration in each PRP (without leukocytes) aliquot was 2 × 10 6 platelets/µL.…”

“…The donors gave their written informed consent to allow the use of PRP for in vitro experimentations for which the Ethical Committee's approval is not required. PRP treatment was performed as previously reported [48,57].…”

“…Fibroblastic cells were induced to differentiate into myofibroblasts by shifting them in differentiation medium (DM) consisting of DMEM supplemented with 2% FBS and 2 ng/mL TGF-β1 (PeproTech, Inc., Rocky Hill, NJ, USA) for 48 h and 72 h, as previously reported [48]. In parallel experiments, to estimate the influence of PRP on fibroblast-myofibroblast transition, PRP was added to DM (1:50) [48,57]. In some experiments, the cells were cultured in PM, DM, or DM + PRP in the presence of 1 mM heptanol (Sigma), a specific GJ blocker, to evaluate the involvement of GJs in myofibroblastic differentiation.…”