Prolyl oligopeptidase induces angiogenesis both in vitro and in vivo in a novel regulatory manner

Abstract

BACKGROUND AND PURPOSE A serine protease, prolyl oligopeptidase (POP) has been reported to be involved in the release of the pro-angiogenic tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Ac-SDKP) from its precursor, 43-mer thymosin β4 (Tβ4). Recently, it was shown that both POP activity and the levels of Ac-SDKP are increased in malignant tumours. The aim of this study was to clarify the release of Ac-SDKP, and test if POP and a POP inhibitor, 4-phenyl-butanoyl-L-prolyl-2(S)-cyanopyrrolidine (KYP-2047), can affect angiogenesis. EXPERIMENTAL APPROACH We used HPLC for bioanalytical and an enzyme immunoassay for pharmacological analysis. Angiogenesis of human umbilical vein endothelial cells was assessed in vitro using a 'tube formation' assay and in vivo using a Matrigel plug assay (BD Biosciences, San Jose, CA, USA) in adult male rats. Moreover, co-localization of POP and blood vessels was studied. KEY RESULTS We showed the sequential hydrolysis of Tβ4: the first-step hydrolysis by proteases to <30-mer peptides is followed by an action of POP. Unexpectedly, POP inhibited the first hydrolysis step, revealing a novel regulation system. POP with Tβ4 significantly induced, while KYP-2047 effectively prevented, angiogenesis in both models compared with Tβ4 addition itself. POP and endothelial cells were abundantly co-localized in vivo. CONCLUSIONS AND IMPLICATIONS We have now revealed that POP is a second-step enzyme in the release of Ac-SDKP from Tβ4, and it has novel autoregulatory effect in the first step. Our results also advocate a role for Ac-SDKP in angiogenesis, and suggest that POP has a pro-angiogenic role via the release of Ac-SDKP from its precursor Tβ4 and POP inhibitors can block this action.