Sub-Toxic Human Amylin Fragment Concentrations Promote the Survival and Proliferation of SH-SY5Y Cells via the Release of VEGF and HspB5 from Endothelial RBE4 Cells.

Giuseppe Caruso,Donatella Distefano,Giacomo Lazzarino,Claudia Fresta,Paolo Parlascino,Susan Lunte,Filippo Caraci,Giuseppe Lazzarino

doi:10.3390/ijms19113659

Abstract

Human amylin is a 37-residue peptide hormone (hA1-37) secreted by β-cells of the pancreas and, along with insulin, is directly associated with type 2 diabetes mellitus (T2DM). Amyloid deposits within the islets of the pancreas represent a hallmark of T2DM. Additionally, amylin aggregates have been found in blood vessels and/or brain of patients with Alzheimer’s disease, alone or co-deposited with β-amyloid. The purpose of this study was to investigate the neuroprotective potential of human amylin in the context of endothelial-neuronal “cross-talk”. We initially performed dose-response experiments to examine cellular toxicity (quantified by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay) of different hA17–29 concentrations in endothelial cells (RBE4). In the culture medium of these cells, we also measured heat shock protein B5 (HspB5) levels by ELISA, finding that even a sub-toxic concentration of hA17–29 (3 µM) produced an increase of HspB5. Using a cell medium of untreated and RBE4 challenged for 48 h with a sub-toxic concentration of hA17–29, we determined the potential beneficial effect of their addition to the medium of neuroblastoma SH-SY5Y cells. These cells were subsequently incubated for 48 h with a toxic concentration of hA17–29 (20 µM). We found a complete inhibition of hA17–29 toxicity, potentially related to the presence in the conditioned medium not only of HspB5, but also of vascular endothelial growth factor (VEGF). Pre-treating SH-SY5Y cells with the anti-Flk1 antibody, blocking the VEGF receptor 2 (VEGFR2), significantly decreased the protective effects of the conditioned RBE4 medium. These data, obtained by indirectly measuring VEGF activity, were strongly corroborated by the direct measurement of VEGF levels in conditioned RBE4 media as detected by ELISA. Altogether, these findings highlighted a novel role of sub-toxic concentrations of human amylin in promoting the secretion of proteic factors by endothelial cells (HspB5 and VEGF) that support the survival and proliferation of neuron-like cells.

Highlights

Human amylin is a peptide hormone composed of 37 amino acids synthesized in β-cells of the pancreas implicated in the regulation of glucose metabolism [1] and co-secreted with insulin in a 1:20 ratio [2]
The results clearly showed that the pre-treatment with anti-Flk1 (Table 1) produced a significant decrease in the protective effects caused by both Conditioned medium (CM)-RBE4 and CM-RBE4-hA17–29, strongly suggesting that the release of vascular endothelial growth factor (VEGF) significantly contributes as the additional protective factor to the overall increased resistance of neuron-like SH-SY5Y cells towards amylin cytotoxic concentrations
Results reported in the present study indicate that human amylin, in concentration not causing change in endothelial cell viability, represents a sufficient stressor to stimulate RBE4 cells in releasing both heat shock protein B5 (HspB5) and VEGF in the extracellular environment

Summary

Introduction

Human amylin (hA) is a peptide hormone composed of 37 amino acids synthesized in β-cells of the pancreas implicated in the regulation of glucose metabolism [1] and co-secreted with insulin in a 1:20 ratio [2]. The “final” full-length peptide (hA1-37) is obtained after the cleavage of its precursor of 89 amino acids in a multi-step process [3,4]. Deposition of hA aggregates has been found in about 90% of patients suffering from type 2 diabetes mellitus (T2DM) [6] and it plays a central role in β-cells death [7]. The same hA deposits, alone or in co-presence of β-amyloid (Aβ), were found in the brain of both Alzheimer’s disease (AD) and T2DM patients [8], showing once again that hA and Aβ represent key factors with common linking pathobiological mechanisms both in AD and TD2M [9]

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