Endocrine Abstracts

Introduction: Although the protective effects of 1,25(OH)₂D₃ on the early stages of atheromatosis process have been investigated, data on its influence on factors implicated in plaque vulnerability are limited. During these stages metalloproteinases MMP-2, MMP-9, their inhibitors TIMP-1,TIMP-2, the RANKL-RANK-OPG system and MCP-1 play a critical role. We aimed to investigate the effect of vitamin D on the expression of the above factors on endothelial cells. Methods: Human aortic endothelial cells (HAECs) were incubated with VitD for 24, 48,72 hours at a concentration range 10⁻⁸ – 10⁻¹¹ in the absence of TNF-α or with VitD for 24 hours in the presence of TNF-α at concentration 10ng/ml for the last 6 hours. The mRNA expression of the aforementioned genes was assessed by real time-PCR. Zymography for MMP-2 and MMP-9 activity was also performed. OPG protein levels were also evaluated by western blot.

Results: VitD did not significantly change the expression of MMP-9, MMP-2, TIMP-1 and TIMP-2 at mRNA level at various incubation times and concentrations. The expression of OPG at mRNA was significantly increased in the presence of TNF-a and this effect became more pronounced when cells were preincubated with Vit D at concentration 10⁻⁹ M. Preincubation with p38 MAP kinase inhibitor totally abolished this result.

When cells activated with TNF-a, preincubation with Vit D resulted in a significant decrease in MMP-9 expression at 10⁻¹⁰ and 10⁻¹¹M and increase in TIMP-2 expression while it didn’t affect the expression of MMP-2 and TIMP-1. Gelatin zymography revealed that Vit D decrease the active MMP-2 dose-dependently.

Conclusions: Vit D at physiologically achievable concentrations reduces the expression of MMP-9 and induces the expression of TIMP-2 at mRNA level implying a possible protective role in the later stages of atheromatosis, such as plaque vulnerability. Further studies are needed to elucidate the underlying mechanisms.