The regulatory role of Insulin-like growth Factor-1 N-terminal tripeptide and diketopiperatzine derivative in Insulin-like growth Factor-1 mediated angiogenesis

Abstract

The N-terminal tripeptide of insulin-like growth factor 1 (IGF-1), glycine-proline-glutmate (GPE), has proven to be neuroprotective in a number of in vitro and in vivo models. However, clinical applications of GPE are limited by poor pharmacokinetic properties, particularly its very short plasma half-life. GPE is metabolised to cyclic glycine-proline (cGP), which is plasma-stable and is postulated to act much more specifically due to its rigid structure. The mechanisms of action of GPE and cGP remain elusive to date. Herein we proposed GPE and cGP to regulate the bioactivity of IGF-1 using angiogenesis as the bioactive platform. Human microvascular endothelial cell line-1 (HMEC-1) was cultured in the presence of exogenous GPE, cGP, IGF-1, and combinations thereof, before the ability of HMEC-1 cells to proliferate, migrate, and form capillary-like structures were assayed in vitro. In addition, the gene expression of IGF-1, IGF-1 receptor (IGF-1R) and binding proteins 2 and 3 (IGFBP-2, -3), and vascular endothelial growth factor (VEGF) in response to acute exposure to GPE and cGP were investigated using real-time quantitative PCR. Our study demonstrated cGP to inhibit IGF-1-induced tube formation and proliferation of HMEC-1 cells in a concentration-dependent manner. The effect on HMEC-1 proliferation was also dependent on time whereby cGP’s inhibitory effect became only apparent after eight days of incubation at earliest. In contrast, GPE was ineffective in regulating the angiogenic actions of IGF-1 in HMEC-1 cells. GPE per se was inhibitory to HMEC-1 cell proliferation inasmuch as cGP, with the onset of effect occurring after eight days of incubation. Acute exposure to cGP (100 nM) did not affect gene expression levels of all genes investigated. In contrast, acute exposure to GPE (100 pM) led to a significant downregulation of IGF-1R and VEGF-A mRNA levels. GPE and cGP are potentially anti-angiogenic through their ability in reducing the growth of HMEC-1 cells. More importantly, cGP may potentially regulate the bioactivity of IGF-1 as evidenced in its inhibitory effect towards IGF-1-induced tube formation and proliferation. GPE failed to influence the angiogenic activity of IGF-1 however the possibility that GPE may affect other bioactivities of IGF-1 remains to be addressed.