Supervisors info:
Ράλλης Μιχαήλ,
Επίκουρος Καθηγητής του Τομέα Φαρμακευτικής Τεχνολογίας, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
The main purpose of the present study was the in vitro biological evaluation of peptides, thymosin α1 (Tα1), thymosin β4 (Τβ4) and its N-terminal tetrapeptide Ac-SDKP using cell strains which are associated with dermal wound healing. The Τα1, Tβ4 and Ac-SDKP belong to the family of thymosins, which are endogenous peptides and are present in almost all human cells.
Both Tβ4 and AcSDKP have a positive effect on the wound healing process of various tissues (heart, kidney, liver, skin) according to in vitro assays (endothelial cells, fibroblasts of cardiac and renal origin etc.) and in vivo assays in animal models. The few studies on the effect of Tα1 on the dermal wound healing phenomenon are mainly related to its angiogenic effect in in vitro assays, as well as, to its overall effect in animal models.
In an attempt to unravel the mechanisms of the healing effects of Tβ4 and Ac-SDKP and to further investigate the potential effects of Tα1, human cutaneous fibroblasts, which are actively participating in most stages of healing, were selected as a study system for an in vitro simulation of the dermal healing process. In particular, the effects of the Tα1, Tβ4 and Ac-SDKP peptides on fibroblast proliferation and migration were studied. Moreover, their effects on the regulation of collagen, matrix metalloprotease (MMP) and tissue inhibitor of MMP (TIMP)gene expression were examined, as well as, on the levels of MMP secretion in fibroblast conditioned media. Finally, the ability of the three thymosinic peptides to trigger pivotal signal transduction pathways was also tested.
Fibroblast proliferation was marginally yet statistically significantly stimulated by all three thymosinic peptides. Migration of these cells was also induced by these peptides, with Tα1 exhibiting the most potent effect. Tβ4 was found to induce both type-I and type-III collagen gene expression, while Tα1 and Ac-SDKP at high concentration (10 nM) inhibited the expression of collagen type-III. All three peptides inhibited MMP-1 gene expression but they stimulated the transcription of MMP-2 gene. TIMP expression was not altered, with the exception of TIMP-1 inhibition by Ac-SDKP. Furthermore, Tα1 and Ac-SDKP were found to induce the secretion of pro-MMP2 in human fibroblast supernatatnts.
Signal transduction studies offered indications of ERK and JNK phosphorylation, especially at the early time-points following their administration. Finally, during simultaneous treatment of human skin fibroblasts with the thymosinic peptides and the potent ECM inducer TGF-β, Smad-3 phosphorylation was found to be enhanced (compared to the individual effect of TGF-β) in a time-dependent manner, a fact consistent with the additive effects exerting the three thymosinic peptides and TGF-β on collagen synthesis when administered jointly.
In coclusion, the results of our in vitro studies support the beneficial effects of the three thymosinic peptides on wound healing through the induction of skin fibroblast proliferation and migration. Also, our data provide some clues regarding the mechanisms underlying the stimulatory effects of the thymosinic peptides on skin fibroblast collagen synthesis.
Keywords:
Fibroblast, Thymosins, Tα1, Τβ4, Αc-SDKP, Wound Healing, TGF-beta, MMPs
