Endocrine Abstracts

Introduction: Malnutrition in neonates and children has been associated with adult-onset diseases such as obesity and atherosclerosis. Infants breastfeeding exhibit a different pattern of orexin-α secretion compared to formula-fed or infants on parenteral feeding. Given the protective role of maternal milk in the onset of metabolic syndrome and atherosclerosis in later life, we hypothesized that orexin-α may be involved in this process. We studied in vitro the effect of orexin-α on cell proliferation and expression of factors involved in atherosclerosis process in human aortic endothelial cells (HAECs).

Methods: HAECs were incubated with orexin-α at concentrations of 40 ng/ml, 200 ng/ml and 400 ng/ml for 6, 12, 24 and 48 hours. MTS assay was performed to evaluate cell viability/proliferation. The expression of MCP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA was measured by real-time qPCR. Orexin-α receptor and MMP-2 protein expression was evaluated by Western blot. Finally, the expression of TIMP-1 protein was studied using an enzyme immunoassay.

Results: Western blot analysis in HAEC cells, revealed very low orexin-α receptor protein level. Incubation of cells with orexin-α for 24 h induced a dose-dependent decrease in TIMP-1 protein expression (200 ng/ml and 400 ng/ml, P<0.05) with stronger suppression exerted at the lowest concentration (40 ng/ml-P<0.01). However, this was not demonstrated at mRNA level. Incubation of HAECs with orexin-α for 6 h resulted in a decrease in MMP-2 expression at mRNA level (all concentrations-P<0.05), and dose-dependently at protein level (400 ng/ml-P<0.05), while incubation of cells for 24 h increased dose-dependently MMP-2 mRNA (400 ng/ml-P<0.05) and protein levels (200 ng/ml P<0.05 and 400ng/ml, P<0.01). Incubation of HAECs with orexin-α at the highest concentration of 400 ng/ml for 6 and 12 h decreased significantly MCP-1 mRNA levels (P<0.05), while incubation for 24 and 48 hours promoted a significant increase of MCP-1 expression (P<0.05). Incubation of cells with orexin-α (400 ng/ml-6h) resulted in significant reduction (P<0.05) of MMP-2/TIMP-1 ratio while this ratio was significantly induced (P<0.01) after 24 h of incubation.

Conclusion: According to our findings, orexin-α may have an equivocal role in atherosclerosis process/plaque stability with its effects depending mainly on incubation duration. Short incubation period of 6 or 12 hours could promote beneficial effects exerted via reducing the expression of MCP-1 and MMP-2/TIMP-1 ratio. In contrast, longer incubation duration of 24–48 hours could result in detrimental effects, by inducing factors involved in atherosclerosis process and plaque instability (MCP-1, MMP-2/TIMP-1). Further investigation is warranted in order to shed light in the mechanism of action of orexin-α in human aortic endothelial cell.