CHAPTER 5
CONCLUSION AND DISCUSSION
These studies establish that exogen S1P promotes the rapid and reversible expression of both S1P3 receptor protein and mRNA in endothelial progenitor cells (Figs 4c and 7). This induction is not concentration-dependent but only a dose seems to be optimal. Further S1P3 receptor induction improved in vitro migration capability of EPC.(Figs-8 a,b)
S1P plays a crucial role in the cardiovascular system: it acts as a proangiogenic molecule, [133] improves endothelial function, [134] myocardial perfusion, [135] and is involved in cardiovascular development. [91,136] Additional studies demonstrate that S1P modulates homing and trafficking of cells. [137,138] In accordance, the present study documents that S1P influences the functional activity of endothelial progenitor cells to improve their migration capability.
S1P exerts its effects by activating the G protein– coupled receptors (GPCRs) S1P1–5. The S1P GPCRs display considerable complexity in their signalling functions, showing both redundancy and functional antagonism (reviewed in [120]). S1P1 appears to only couple to Gi proteins to activate a number of signaling pathways, including ERK1/2 to enhance cell proliferation, Akt to enhance survival, and Rac to enhance migration [82,121,123]. S1P3 couples to Gi, Gq and G12 proteins, to activate ERK1/2, PLC, Akt, Rho and Rac [82,121,123]. S1P2 also couples to Gi, Gq and G12 proteins, and like S1P1 and S1P2 stimulates ERK1/2, activates Rho but also inhibits Rac, thus promoting stress fibre formation and inhibiting cell migration [82,121,123].
S1P1–3 are widely expressed and have been shown to be involved in the development of a mature vascular system in embryos. S1P1 and S1P3 are considered to be the major receptors in endothelial cells contributing to angiogenic processes.
All data present in literature are regarding mature endothelial cells while only a study exist on progenitor cells; for this reason we have decided to investigate these receptors in EPC. In this
regard our study demonstrates that all S1P receptors are expressed on EPC but with S1P1 and S1P3 at low and very low levels respectively .
Since S1P1 and S1P3 are responsible for the endothelial cell mobility, our hypothesis is that EPC do not migrate in basal conditions. So we wanted to evaluate if a stimulation for these receptors expression could be crucial.
In this regard S1P3 receptor stimulation by S1P seemed to improve the progenitor cells migration in vitro, even if this improvement was not statistically significant respect to the basal level. Different explanations could be given for these findings i.e 1) S1P3 is not specifically required for the EPC migration, 2) S1P1 and S1P3 have been shown to act cooperatively in mature endothelial cells, [139] but S1P had no effect on S1P1 receptor expression or 3) other signalling mechanisms such as relative to S1P2 could be involved.
Indeed we have demonstrated that S1P2 is expressed at significantly high levels on EPC. Based on these data we speculate that it could be responsible for the not significant migration, independently from S1P3 expression. In this regard using a selective S1P2 receptor antagonist, JTE-013, both in presence or absence of S1P, we demonstrated that only inhibition of S1P2 increased the number of EPC migrated and that no synergic effect between JTE-013 and S1P was present. In our opinion this could suggest that the high expression of S1P2 rather than the low expression of S1P3 is involved in the poor mobility of EPC.
Another original result of this study is about VEGF. VEGF in our model acts as S1P and promotes the rapid expression of both S1P3 receptor mRNA and protein in EPC (Figs.6 and 7) at a physiological concentration. The VEGF-induced increase in S1P3 receptor expression leads to enhanced cellular responses (migration) to subsequent S1P treatment.
In other experimental setting we have also investigated the modulation of VEGF by S1P. In contrast to the highly significant expression of S1P3 induced by VEGF, we found that the treatment with S1P at different times had not any effect on VEGF expression in EPC. Although diverse growth factors exist that might influence transcriptional pathways in progenitor endothelial cells, and in
contrast to studies showing that VEGF is a powerful inductor of S1P1 in endothelial cells [140], our results suggest that VEGF may be unique in its ability to induce S1P3 expression in this experimental system. VEGF-induced S1P3 expression is seen within 30 min of VEGF addition. This is a strikingly rapid time course for induction of G protein-coupled receptors in response to an extracellular signal and suggests that S1P3 receptor expression may be subject to dynamic regulation by VEGF under (patho)physiological conditions. So like S1P, also VEGF determines an increase into migration of EPC but a synergic effect with S1P seems not to be present (Fig.8b.). Furthermore, the simultaneous inhibition of S1P2 receptor, responsible for inhibition of migration, didn’t change (Fig.8c) the result, so two different mechanisms could exist for S1P and VEGF.
Because VEGF seems to potentiate S1P-mediated responses through induction of S1P3 expression, and given that the principal VEGF receptor in endothelial cells, is KDR, the proposal [141] that S1P elicits its cellular responses through KDR activation is plausible. However, our results show that KDR expression is very low at basal level and S1P doesn’t induce it. So rather than S1P3 responses being mediated by KDR transactivation, as proposed for S1P1 [141], we suggest that VEGF-mediated S1P3 induction could serve to explain the principal form of ‘‘cross-talk’’ between these two important signaling pathways.
However, KDR is a receptor tyrosine kinase that also modulates several distal serine-threonine protein kinases, so the intracellular signaling pathways that lead to S1P3 induction should be explored by using pharmacological approaches with a series of protein kinase inhibitors to understand the regulatory mechanisms whereby VEGF induces S1P3 expression.
Thus, based on our current observations, it is intriguing to speculate that VEGF-mediated induction of S1P3 protein could represent a mechanism for the synergistic activation of signaling pathways elicited by G protein coupled S1P-EDG and tyrosine kinase-coupled VEGF pathways in progenitor endothelial cells and ultimately exerting actions to modulate angiogenesis.
The understanding the role of S1P in the regulation of many important physiological and pathophysiological processes in EPC, may provide opportunities of future therapeutics. Indeed
recent data indicate that the therapeutic success is determined by functional properties of transplanted cells, [38,39] providing the basis for improvement of functional activities, eg, by pharmacological stimulation of surface receptors in order to enhance homing of progenitor or stem cells, in patients with cardiovascular diseases.
The future challenge remains, however, to understand how the multifaceted interplay between the signalling pathways regulated by sphingolipids is integrated in the complex nature of progenitor cell biology.
