• migration
• EPC
• Sphingolipids

Background Endothelial progenitor cells (EPC) are believed to be the postnatal equivalent of angioblasts, i.e. those cells found in embryonic life which form the walls of blood island that gradually develop into vasculature. Alongside an understanding of potential therapeutic applications, there has also been increasing appreciation of the physiological roles that these cells might play in the homesostasis of the adult vasculature.
Autologous transplantation of culture-expanded EPC successfully promotes therapeutic neovascularization in both ischemic hind limbs as well as acute myocardial infarction models. 2–6
Mechanistically, these cells can either induce angiogenesis by incorporation into vascular structures depicting phenotypes of endothelial cells or may induce angiogenesis by production of growth factors acting in a paracrine manner
However, EPC derived from patients are functionally impaired compared with EPC from healthy donors. Recent data indicate that the therapeutic success is determined by functional properties of transplanted cells, providing the basis for improvement of functional activities, eg, by pharmacological stimulation of surface receptors in order to enhance homing .
One important family of surface receptors is the family of the S1P receptors. These receptors bind sphingosine 1-phosphate (S1P), a bioactive sphingolipid, that has been demonstrated to play a crucial role in the cardiovascular system modulating essential cellular processes including cell growth and survival, regulation of cell motility and invasion, angiogenesis and vascular maturation lymphocyte trafficking and immune regulation. Many agents that have been examined to increase EPC and enhance their function, but only recently some sphingolipids, are becoming intriguing. Accumulated evidence has demonstrated that sphingolipids mediates pro-angiogenic activities, such as endothelial cell proliferation, chemotaxis, and vessel morphogenesis and their actions are mediated by the G-protein-coupled receptors of the endothelial differentiation gene (EDG) family. The physiological and pathophysiological actions of sphingolipids in the regulation of human endothelial progenitors cells are still to be fully realized.
Aim To evaluate the expression of S1P receptors on human EPC, isolated from peripheral blood and to provide experimental evidence that S1P treatment induces pro-angiogenic signalling on EPC.
Materials and Methods “Early” EPC were obtained from mononuclear cells isolated from peripheral blood by seeding on fibronectin-coated dishes (41x106 cells/dish). At first aim we evaluated S1P receptors expression at basal level by using Real time PCR. HUVEC were used as positive control.
Cell viability after 5 days of culture was evaluated by WST-1 assay in presence of different concentrations of S1P and compared with viability of cells cultured in absence of S1P.
In order to establish the effect of S1P treatment on the S1P receptors mRNA level, after 5 days, cells were treated with different concentrations of S1P (0.001-10 μmol/L) for 24 h. In another set of experiments different time points (2, 4, 6, 8 hours) of treatment were taken in account incubating cells with 100 nM S1P. Moreover VEGF expression was also investigated in presence or in absence of S1P (0.001-10 μmol/L). S1P receptors were also analyzed after VEGF (50 ng/ml) treatment of EPC for 30 minutes. In addition the effect of S1P on directed migration and tube formation on matrigel with and without VEGF were also evaluated.
Results Amplified corresponding to mRNA transcripts encoding S1P1-5, were detected by real time PCR except for S1P3. The analysis revealed that the relative mRNA abundance in EPCs was S1P2 > S1P5 >S1P4 >S1P1. In addition S1P2 and S1P5 mRNAs were significantly higher in EPC when compared to HUVEC where S1P3 was the most expressed. Cell viability after 5 days of culture showed no significant difference after treatment with S1P at tested doses compared to untreated cells. The treatment of EPC with different doses of lipid showed no significant effect on the receptors expression except for S1P3 at 100nM dose. On the contrary, it seemed that a time-dependent response was present, because all S1P receptors were upregulated at 2h but a significant upregulation was observed at 2h only for the S1P3 and S1P5 receptors, while a significant downregulation for S1P2 at 4 and 6 h was observed when compared to basal.
VEGF was expressed at basal conditions but at a very low level; S1P treatment at different concentrations and at different time points (with S1P 100 nM), had no significant effect on its expression. The converse experiment where cultured EPC were treated with VEGF (50 ng/ml), the expression of S1P3 mRNA increased by approximately 50-fold compared to basal level. This up-regulation by VEGF was seen within 30 min of VEGF addition. By contrast, expression of remaining receptors was not altered by VEGF treatment.
The S1P3 expression after treatment with S1P (100 nM, 2h) or VEGF (50 ng/ml, 30 min) was also confirmed by western blot.
We observed a dose-dependent chemotactic effect of S1P on untreated EPC with a maximal response when 100 nM S1P was added to the lower chamber of the migration system. However these results were not statistically significant when compared to basal or to the VEGF-induced migration. The result didn’t change when EPC was pre-incubated with S1P or VEGF. In addition the synergic effect between VEGF and S1P was not observed. When EPC were pre-incubated with JTE-013 alone, the migratory capability was increased compared to basal EPC whereas the simultaneous presence of the inhibitor and S1P or VEGF didn’t modify the mobility of EPC.
Conclusions Our results indicate that a specific regulation of S1P-modulated functional responses in EPC could be achieved by S1P receptors agonist and that manipulation of S1P biological activities may be applied in clinical progenitor cell therapy to improve EPC function in patients with cardiovascular diseases.