Differential profile of protein expression on human keratocytes treated with autologous serum and plasma rich in growth factors (PRGF)

Abstract

PurposeThe main objective of this study is to compare the protein expression of human keratocytes treated with Plasma rich in growth factors (PRGF) or autologous serum (AS) and previously induced to myofibroblast by TGF-β1 treatment.MethodsBlood from healthy donor was collected and processed to obtain AS and PRGF eye drops. Blood derivates were aliquoted and stored at -80°C until use. Keratocyte cells were pretreated for 60 minutes with 2.5 ng/ml TGF-β1. After that, cells were treated with PRGF, AS or with TGF-β1 (control). To characterize the proteins deregulated after PRGF and AS treatment, a proteomic approach that combines 1D-SDS–PAGE approach followed by LC–MS/MS was carried out.ResultsResults show a catalogue of key proteins in close contact with a myofibroblastic differentiated phenotype in AS treated-cells, whereas PRGF-treated cells show attenuation on this phenotype. The number of proteins downregulated after PRGF treatment or upregulated in AS-treated cells suggest a close relationship between AS-treated cells and cytoskeletal functions. On the other hand, proteins upregulated after PRGF-treatment or downregulated in AS-treated cells reveal a greater association with processes such as protein synthesis, proliferation and cellular motility.ConclusionThis proteomic analysis helps to understand the molecular events underlying AS and PRGF-driven tissue regeneration processes, providing new evidence that comes along with the modulation of TGF-β1 activity and the reversion of the myofibroblastic phenotype by PRGF.

Proteomic Characterization of Plasma Rich in Growth Factors and Undiluted Autologous Serum

“…Finally, a recent study showed that PRGF eye drops were able to produce a significant reduction in the number of transforming growth factor (TGF)-β1-induced myofibroblasts in comparison to AS, suggesting that PRGF may promote corneal wound healing regeneration, reducing scar formation [15]. To understand the pathways by which PRGF exerts its antifibrotic potential in comparison to AS, a proteomic study was performed in HK cells differentiated to myofibroblasts, which previously were to be treated with PRGF or AS [23]. This study shows that PRGF treatment inactivated or reduced the activation of several proteins involved in the pathways, whereby TGF-β1 exerts its action to induce the formation of α-actin fibers on human corneal stromal keratocytes (HK), inducing their transformation to myofibroblasts.…”

<p>Plasma Rich in Growth Factors for the Treatment of Cicatrizing Conjunctivitis</p>

“…Indeed, the different PRGF-based formulations including injectable liquid, eye drops, clot and membrane (for outpatient treatment and surgical use) exert regenerative, anti-inflammatory, antifibrotic, anti-bacterial, proliferative and cell migration properties. [14][15][16] Therefore, CC therapy should be focused on regeneration and on avoiding inflammation and fibrosis before reaching the risk of corneal blindness or other ocular surface complications. In this particular study, and intending to avoid the potential risk of certain side effects related to the PRGF's IgE levels and complement activity in autoimmune diseases or other refractory cases to the treatment, we decided to apply Immunosafe PRGF formulations.…”

“…The protocol for the preparation of Endoret-PRGF eye drops (ePRGF) is described in the kit's instructions for use, and it has been published by Anitua and Col. 15,16,20 The procedure was carried out under sterile conditions in the surgical room. To prepare the ePRGF, 70 mL of peripheral blood was collected into 9 mL tubes with 3.8% sodium citrate as an anticoagulant.…”

“…13 PRGF also increases the proliferation and migration activity of corneal keratocytes and conjunctival fibroblasts, reducing the TGFβ-1 activity, leading to a tissue regeneration approach that limits the fibrosis pathways. [14][15][16] It has been reported that immunosafe PRGF (isPRGF) treatment, which implies a 1-hour extra heat treatment at 56º C, preserves the content of most of the proteins and morphogens involved in the wound healing process while reduces the content of IgE and complement activity drastically. 20 In diseases related to an immune system unbalance, and an acute inflammatory response such as Sjögren Syndrome and GVHD, the effect of isPRGF has been evaluated with satisfactory results.…”

Plasma rico en plaquetas como terapia autóloga en la medicina regenerativa: Revisión narrativa

“…El PRP es un hemoderivado con alta concentración de plaquetas, que puede servir para regeneración celular y activación biológica de diversos sistemas para responder a procesos traumáticos, degenerativos o infecciosos. Los factores de coagulación y citocinas están almacenados principalmente dentro de los gránulos alfa (Castro-Piedra & Arias-Varela, 2019), que son los responsables de inducir diferentes respuestas biológicas en la hemostasias y regeneración, constituyendo también el plasma rico en factores de crecimiento (PRFC) (Alcazar-Rubio et al, 2015;Anitua et al, 2018) (Tabla 1). La capacidad de cicatrización, regeneración y defensa está modulada por las células mesenquimales como células madre que dan origen a la mayoría de las células (Hersant et al, 2019).…”