Author + information
Currently, it is demonstrated that ghrelin has potently cardiovascular protective effects in diabetes. The aim of this research was to investigate the role of octanoylated ghrelin in regulating diabetic vascular calcification and the underlying mechanisms.
In vivo investigation, we consecutively enrolled thirty cases of type 2 diabetes patients with foot amputation from the Affiliated Hospital of Jiangsu University from January 2013. The patients exhibited increased vascular calcification, which were divided into three groups based on the tibial artery stenosis of Doppler ultrasound: mild stenosis group (0%<stenosis <50%)(n=10), moderate stenosis group (50% ≤ stenosis <70%)(n=10) and severe stenosis/occlusion group (70% ≤ stenosis ≤ 100%)(n=10). Anthropometric data such as serum indicators were measured using enzyme-linked immune sorbent assay (ELISA). In vitro investigation,rat vascular smooth muscle cells (VSMCs) were incubated with high-glucose(HG,25mmol/L D-glucose),high-lipid(HL,palmitic acid 600umol/L) and β-glycerophosphate (β-GP, 10 mmol/L) and then treated with various concentrations of octanoylated ghrelin (10?8M∼10?5M). The total calcium content, alkaline phosphatase activity (ALP) and western blot analysis were then performed.
1. In vivo study, morphological analysis showed diffuse calcium deposition in the anterior tibial artery of diabetic amputee, but compared with mild and moderate stenosis group, spotty calcification in the atherosclerotic plaques of severe stenosis/occlusion group was more significantly (hard hyperechoic plaque in ultrasound),as well as the quantitative tests of the ALP activity and calcium content in tibial artery. With the aggravation of vascular stenosis,western blot analysis indicated the expression of osteoprotegerin(OPG) and ghrelin were down-regulated and the expression of receptor activator of nuclear factor kappa B ligand (RANKL) was up-regulated. Pearson correlation analysis revealed a negative correlation between calcium content of artery wall and ghrelin levels (r = -0.63, P<0.001) as well as between ghrelin levels and sRANKL levels (r =-0.72, P<0.001),while OPG levels were positively correlated with ghrelin levels (r = 0.77 P<0.001).
2. In vitro study, HG, HL and β-GP induced a significant increase in VSMCs total calcium content, ALP activity and expression of osteogenic marker.Octanoylated ghrelin at 10?8M∼10?5M blunted the calcification in VSMCs, with decreased total calcium content, ALP activity, RANKL expression and increased OPG expression, in a dose-dependent manner. In addition, when VSMCs were pretreated by anti-OPG antibody, anti-RANKL antibody, ghrelin treatment also suggested a corresponding role in VSMCs calcification.
Serum octanoylated ghrelin levels may be a predicator of the vascular calcification in diabetes mellitus. The possible mechanism of ghrelin attenuating vascular calcification in diabetes mellitus is related with OPG/RANKL signal.