RESUMO
INTRODUCTION: Preeclampsia is multifactorial in origin but the primary trigger is thought to be related to impaired placentation which is followed by systemic maternal responses. Vitamin D3 deficiency is a worldwide problem and is associated with a substantial increase in preeclampsia risk. Endothelial progenitor cells, in particular their highly proliferative subpopulation of endothelial colony forming cells (ECFC), play an important role in placental vasculogenesis and endothelial repair capacity. However, the mechanisms of vitamin D3 influence on placental development are poorly understood. OBJECTIVES: Therefore we investigated the influence of vitamin D3 on the differentiation of endothelial progenitor cells (ECFCs) in a placental angiogenesis model and hypothesized that vitamin D3 stimulates the expression of vascular endothelial growth factor (VEGF) in ECFCs. METHODS: Umbilical cord blood was obtained from uncomplicated, term pregnancies, the mononuclear cells were isolated and seeded onto collagen-coated culture plates for outgrowth of ECFCs. After preincubation with 10 nM vitamin D3, ECFCs were plated onto Matrigel (BD Biosciences) in the presence of the treatment media. After 6 hours capillary-like tubules were fixed and their total length was determined per well and median values were calculated from n=38 experiments. For mRNA expression analyses total RNA isolation was performed. High capacity cDNA reverse transcription kit (Invitrogen) was used for cDNA synthesis and Real time RT-PCR was performed on the Rotor Gene 6000 PCR instrument (Corbett Research) using VEGF-A primers according to existing literature. Statistical analysis was performed using Wilcoxon signed rank test. RESULTS: Our experiments show a significant promoting effect of vitamin D3 on tubule formation in ECFCs. ECFCs treated with 10nM vitamin D3 showed a 1.27 times higher tubule formation compared to vehicle-treated controls (1.27±0.19, p<0.05, n=38). mRNA expression analysis showed a 1.8 times higher expression of VEGF-A mRNA in ECFCs treated with 10nM vitamin D3 compared to controls (1.82±0.43, p<0.0001, n=18). CONCLUSION: Physiological concentrations of vitamin D3 significantly promote the formation of capillary-like structures by ECFCs in a cell culture model. This effect is mediated by an up-regulation of VEGF-mRNA in ECFCs by Vitamin D3. Since the de novo angiogenesis is a crucial step in development of the placenta, a vitamin D deficiency could play an important role in the pathophysiology of preeclampsia. This finding goes along with clinical studies in which vitamin D deficiency increases the risk of preeclampsia substantially.
