Immunomodulator FTY720 Induces eNOS-Dependent Arterial Vasodilatation via the Lysophospholipid Receptor S1P ₃

Abstract

Abstract-The novel immunomodulator FTY720 is effective in experimental models of transplantation and autoimmunity, and is currently undergoing Phase III clinical trials for prevention of kidney graft rejection. FTY720 is a structural analogue of sphingosine-1-phosphate (S1P) and activates several of the S1P receptors. We show that FTY720 induces endothelium-dependent arterial vasodilation in phenylephrine precontracted mouse aortae. Vasodilation did not occur in thoracic aortic rings from eNOS-deficient mice, implicating and effect dependent of activation of the eNOS/NO pathway. Accordingly, FTY720 induced NO release, Akt-dependent eNOS phosphorylation and activation in human endothelial cells. For biological efficacy, FTY720 required endogenous phosphorylation, since addition of the sphingosine kinase antagonist NЈ,N-dimethylsphingosine (DMS) prevented activation of eNOS in vitro and inhibited vasodilation in isolated arteries. The endothelial phosphorylation of FTY720 was extremely rapid with almost complete conversion after 10 minutes as determined by mass spectrometry. Finally, we identified the lysophospholipid receptor S1P 3 as the S1P receptor responsible for arterial vasodilation by FTY720, as the effect was completely abolished in arteries from S1P 3 -deficient mice. In summary, we have identified FTY720 as the first immunomodulator for prevention of organ graft rejection in clinical development that, in addition, positively affects the endothelium by stimulating NO production, and thus potentially displaying beneficial effects on transplant survival beyond classical T cell immunosuppression. Key Words: FTY720 Ⅲ eNOS Ⅲ S1P receptor T he novel immunomodulator FTY720 is currently undergoing Phase III clinical trials for prevention of kidney graft rejection. 1 FTY720 shares striking structural homology to sphingosine 1-phosphate (S1P), 2 a natural lysophospholipid that is present at high nanomolar (nmol/L) concentrations in serum. 3 Recent data show that FTY720 is phosphorylated in vivo by sphingosine-kinase-2 (SphK2), 4 and that the FTY720-phosphate metabolite (FTY720-P) is a potent agonist of 4 of the 5 G protein-coupled receptors for S1P: S1P 1 , S1P 3 , S1P 4 , and S1P 5 . 2,5 Recent studies show that the S1P 1 receptor and its natural ligand S1P are pivotal to lymphocyte recirculation: mice with a specific deletion of S1P 1 in hematopoietic cells showed that thymocytes selectively require S1P 1 for egress from thymus, whereas both T and B cells require this receptor for egress from peripheral lymphoid organs. 6 Thus, it was suggested that the efficacy of FTY720 in transplantation and autoimmunity may relate primarily to an inhibition of effector T cell recirculation from lymphoid organs to peripheral sites of inflammation. S1P receptor agonists mediate a variety of physiological processes and stimulate multiple signaling pathways resulting in calcium mobilization from intracellular stores, polymerization of actin, chemotaxis/migration, and escape from apoptosis. 7-10 S1P is released by platelets durin...

Sphingosine Kinase–Dependent Activation of Endothelial Nitric Oxide Synthase by Angiotensin II

“…With the use of S1P 3 knockout mice, for example, it was recently shown that HDLs, known to carry S1P, and the immunomodulator and S1P receptor agonist FTY720 induce an endothelium-and NO-dependent vasorelaxation via the S1P 3 receptor in vitro and ex vivo. 34,42 Taken together, these data suggest that activation of the endothelial AT 1 receptor by Ang II leads to a modulation of the sphingolipid metabolism, resulting in increased NO production. This is most likely the result of increased sphingosine kinase activity leading to increased production of S1P that subsequently stimulates (an) endothelial S1P receptor(s).…”

Sphingosine-1-phosphate and modulation of vascular tone

“…74 A recent study reported that FTY-720 mediates eNOS-dependent vasorelaxation in phenylephrinepreconstricted mouse thoracic aorta preparations by way of S1P 3 receptor activation. 75 Collectively these studies help establish that treatment with S1P leads to physiologically relevant amounts of NO production in intact blood vessels.…”

The S1P1 receptor-selective agonist CYM-5442 reduces the severity of acute GVHD by inhibiting macrophage recruitment

“…45 An S1P 1 -selective agonist may act directly on macrophage proliferation and trafficking. [26][27][28][29]46 An in vivo proliferation assay demonstrated that CYM did not affect macrophage proliferation. Increasing amounts of data have indicated that the lower number of macrophages in the inflamed tissues of FTY720-treated mice could be an indirect effect of vascular permeability and monocyte recruitment.…”

“…CYM (3 mg/kg), 5,27,28 control water, or FTY720 was intraperitoneally injected daily from day 21 to day 3 post allogeneic bone marrow transplantation (BMT). Ten mice per group were monitored daily for survival, were weighed every 3 days, and were thereafter examined for the clinical characteristics of aGVHD.…”

“…S1P might also be involved in macrophage trafficking. 3,[26][27][28][29] FTY720 reduces macrophage infiltration into sites of inflammation in many inflammatory disease models. [30][31][32][33] The mechanism of action of FTY720 may involve its effects on endothelial cells to reduce monocyte migration.…”