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. Author manuscript; available in PMC: 2018 Aug 1.
Published in final edited form as: Hypertension. 2017 Jun 12;70(2):426–434. doi: 10.1161/HYPERTENSIONAHA.117.09088

S1PR1 signaling regulates blood flow and pressure

Anna Cantalupo 1, Antonella Gargiulo 1,2, Elona Dautaj 1, Catherine Liu 1, Yi Zhang 1, Timothy Hla 1, Annarita Di Lorenzo 1,*
PMCID: PMC5531041  NIHMSID: NIHMS876641  PMID: 28607130

Abstract

Nitric oxide is one of the major endothelial-derived vasoactive factors that regulate blood pressure and the bioactive lipid mediator S1P is a potent activator of endothelial nitric oxide synthase through G-protein coupled receptors. Endothelial-derived S1P and the autocrine/paracrine activation of S1P receptors play an important role in preserving vascular functions and blood pressure homeostasis. Furthermore, FTY720, binding to four out of five S1PRs recently approved by the FDA to treat autoimmune conditions, induces a modest and transient decrease in heart rate in both animals and humans, suggesting that drugs targeting sphingolipid signaling affect cardiovascular functions in vivo. However, the role of specific S1P receptors in BP homeostasis remains unknown.

The aim of this study is to determine the role of the key vascular S1P receptors, namely, S1PR1 and S1PR3 in BP regulation in physiological and hypertensive conditions. The specific loss of endothelial S1PR1 decreases basal and stimulated endothelial-derived NO, and re-sets blood pressure to a higher-than-normal value. Interestingly, we identified a novel and important role for S1PR1 signaling in flow-mediated mechanotransduction. FTY720 (fingolimod), acting as functional antagonist of S1PR1, markedly decreases endothelial S1PR1, increases blood pressure in control mice and exacerbates hypertension in Ang-II mouse model, underlining the anti-hypertensive functions of S1PR1 signaling.

Our study identifies S1P-S1PR1-NO signaling as a new regulatory pathway in vivo of vascular relaxation to flow and blood pressure homeostasis, providing a novel therapeutic target for the treatment of hypertension.

Keywords: S1P receptors, Flow, Vascular tone, Hypertension

INTRODUCTION

In spite of the available number of anti-hypertensive agents, BP management remains difficult for many patients and leads to a progressive end organ dysfunction, suggesting the need for alternative therapeutic options.

The bioactive lipid, S1P has emerged as an important regulator of vascular development and stability1, 2, angiogenesis3, permeability4 and ex-vivo vascular tone5, 6. These cardiovascular functions are mediated by three out of the five G-protein coupled receptors, named S1PR1-3. In the endothelium, S1PR1 is highly abundant whereas S1PR3 is expressed at a lower level4. S1P strongly activates eNOS-derived NO production5, comparable in magnitude to VEGF and bradykinin7. Recently, Cantalupo et al. discovered an important role of endothelial-derived S1P in blood flow and pressure regulation through the autocrine S1PR1/eNOS signaling and demonstrated that up-regulation of endothelial-derived S1P prevents the onset of hypertension6. Furthermore, SEW2871, an agonist of S1PR1, restores normal BP in hypertensive mice6 and prevents the onset pathological cardiac hypertrophy8, revealing important cardiovascular protective functions of the S1PR1 signaling.

S1PR3 is expressed in vascular smooth muscle cells (VSMC) of various vascular beds9, 10, and along with S1PR2 induce vasoconstriction via Gq-coupled Ca2+/IP3 and G13-coupled RhoA/Rho kinase pathway11, 12. On the contrary, endothelial S1PR3 mediates HDL-bound-S1P induced vasodilation of thoracic aorta ex-vivo via eNOS-derived NO production13. Consistently, in vivo, S1P triggers a small and transient decrease in BP in WT but not in S1pr3−/− mice14, suggesting an involvement of S1PR3 in the BP effects of S1P.

Although the roles of S1PR1-3 have been examined during vascular development2, the impact of S1PR1,3-signaling on vascular tone and BP regulation remain poorly understood.

Our study shows that S1PR1 plays a predominant role BP homeostasis, via NO-mediated vasodilation in response to S1P, and particularly to flow, unveiling a novel role of S1PR1 as mechano-transducer. S1PR3 is mainly responsible for S1P-induced vasoconstriction and myogenic tone. FTY720, a prodrug that targets four of the five S1P receptors, S1PR1, S1PR3, S1PR4 and S1PR515, with highest affinity for S1PR116, was approved for use by the FDA for the treatment of relapsing-remitting multiple sclerosis15, 17. When administered chronically, FTY720 induces the internalization and degradation of S1PR1, hence defined “functional antagonist”18. A major biological function of FTY720 is to block the egress of lymphocytes from lymphoid tissues19. The activation of the receptors S1P1 in the cardiovascular system accounts for the transient effects of FTY720 on heart rate, on the atrioventricular conduction and the lasting effect on BP17, 20. Because our findings suggested a critical role for S1PR1 signaling in the BP homeostasis, we also investigated the impact of chronic FTY720 treatment of vascular tone and BP homeostasis. FTY720 treatment increased BP in control mice, exacerbates hypertension and impaired endothelial-dependent vasodilation. These data corroborate the protective role of S1P signaling in BP regulation.

Our study reveals for the first time a critical role of S1PR1-NO signaling in flow-mediated vasodilation and BP homeostasis, and underlines potential deleterious effects of FTY720 on the vascular functions. Furthermore, it identifies specific roles of S1PR1 and 3 in the vasomotor reactivity of resistance arteries to S1P, flow and intraluminal pressure.

METHODS

Animals

S1pr1 floxed mice (S1pr1f/f)21 were crossed to VE-Cadherin-Cre-ERT2 to generate mice lacking S1PR1 specifically in endothelial cells (ECKO-S1pr1). These mice have been backcrossed with C57BL/6 for more than 8 generations. To excise the floxed-S1pr1 gene, mice were treated daily with tamoxifen 3 mg/Kg for five consecutive days at the age of 6–8 weeks. S1pr3−/− mice were generated as previously described2. Heterozygous S1p3 mice were bred to generate S1pr3−/− and control littermates used for this study. The C57BL/6 mice used for the experiments with FTY720 in normotension and hypertension are generated in our laboratory by breeding C57BL/6 mice. All of the studies were performed according to protocols approved by the Weill Cornell Institutional Animal Care and Use Committee.

Statistical analysis

Data were expressed as mean±SEM. One- or two-way ANOVA were used for all statistical analyses except where Student’s t-test analysis was used. Differences were considered statistically significant when P<0.05. All the tests were two-sided. GraphPad Prism software (version 6.0, GraphPad Software, San Diego, CA) was used for all the statistical analysis.

Detailed description of the Materials and Methods is available in the online-only Data Supplement. Please see http://hyper.ahajournals.org.

RESULTS

Endothelial S1PR1, but not S1PR3, mediates S1P- and flow-induced vascular tone and BP homeostasis via eNOS activation

We have previously demonstrated that the inhibition of S1PR1 with W146 suppressed flow-mediated vasodilation, whereas the administration of SEW2871 in hypertensive mice strongly reduced BP to normal values6, suggesting a protective role of S1PR1 in hypertension. To directly assess the role of S1PR1 in vascular tone and BP regulation we used a conditional knockout mouse model lacking S1PR1 specifically in endothelial cells (ECKO-S1pr1)22 and measured BP in vivo and vasomotor reactivity of mesenteric arteries (MA) ex-vivo by using the pressure myograph system6. At baseline, ECKO-S1pr1 systolic BP (SBP) was higher than normal values (Fig. 1A). S1P-mediated vasodilation was markedly reduced in ECKO-S1pr1 MA (Fig. 1Ba), whereas acetylcholine (Ach)-induced vasodilation was preserved (Fig. 1Bb), suggesting that these signaling pathways are not inter-dependent. Interestingly, flow-induced vasodilation was blunted in ECKO-S1pr1 MA, revealing a novel role for S1PR1 in mechanotransduction signaling (Fig. 1Bc). An important mediator of S1P and flow-induced vasorelaxation is eNOS-derived NO (Fig. S1 A–D). ECKO-S1pr1 mice showed a reduction in basal and Ach-stimulated NO production in MA (Fig. 1Ca,b), in NOx plasma levels (Fig. 1Da) compared to control mice, and significantly lower NOx levels in MA (Fig. 1Db).

Figure 1. The loss of S1PR1, but not S1PR3, induces high blood pressure and impairs flow-mediated vasodilation.

Figure 1

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SBP in (A) ECKO-S1pr1 and S1pr1f/f mice, measured with tail-cuff system. n≥8 mice/group. ECKO-S1pr1 and S1pr1f/f MA were assessed by pressure myograph system for (Ba) S1P-induced vasodilation, n≥5 mice/group (n≥8 MA/group); (Bb) Ach-induced vasodilation, n≥7 mice/group (n≥14 MA/group); (Bc) flow-mediated vasodilation, n≥5 mice/group (n≥9 MA/group). (Ca) Δ decrease of the inner diameter induced by L-NG-nitroarginine methyl ester (L-NAME) and (Cb) Ach-response after incubation of ECKO-S1pr1 and S1pr1f/f with L-NAME, n≥4 mice/group (n≥8 MA/group).

Nitrite (NOx) levels were measured in (Da) plasma of ECKO-S1pr1 and S1pr1f/f mice by using the Griess reaction, n≥4 mice/group and (Db) in MA from the same groups by using a fluorimetric assay, n=5 mice/group. (E) SBP in S1pr3−/− and littermate WT mice, n≥8 mice/group measured with tail cuff system. S1pr3−/− and WT MA were assessed for vasodilation to (Fa) S1P, n≥5 mice/group (n≥7 MA/group); (Fb) Ach, n≥7 mice/group (n≥14 MA/group) and (Fc) flow, n≥5 mice/group (n≥9 MA/group). (Fd) S1pr3−/− and WT plasma nitrite (NOx) were assessed by Griess reaction, n=7 mice/group. Data were expressed as the mean ± s.e.m. *P<0.05; **P<0.01 and ***P<0.001 compared to control mice. Statistical significance was determined by unpaired T-test or Two-way ANOVA.

On the contrary the loss of S1PR3 did not alter BP in vivo (Fig. 1E), the vasorelaxation of MA in response to S1P (Fig. 1Fa), Ach and flow (Fig. 1Fb,c) and NOx plasma levels (Fig. 1Fd), suggesting that S1PR3 is dispensable to mediate endothelial-regulation of vascular tone in resistance arteries.

These findings reveal a predominant role of S1PR1 in S1P- and flow-mediated vascular tone and BP homeostasis.

S1PR3 controls vasoconstriction to S1P and pressure of resistance arteries

Vascular studies showed that S1P-mediated vasoconstriction was markedly increased in ECKO-S1pr1 MA (Fig. 2A), most likely imputable to the lack of S1PR1-eNOS-NO pathway activation in the endothelium. On the contrary, the vasoconstriction of S1pr3−/− MA in response to S1P (Fig. 2B) and intraluminal pressure (Fig. 2Ca, Da), suggesting a direct and novel role for S1PR3 in myogenic tone regulation. ECKO-S1pr1, but not S1pr3−/− MA also showed an increased internal diameter, index of outward remodeling of the vessel (Fig. 2Cb). Finally, PE-induced vasoconstriction was not altered in ECKO-S1pr1 and S1p3−/− MA compared to control (Fig. 2E, F). Altogether, these data indicate that VSMC S1PR3 plays a major role in regulating MA tone in response to S1P and pressure, while S1PR1 contributes mainly to the endothelial-dependent regulation of vascular tone and structure.

Figure 2. The lack of S1PR3 specifically decreases the vasoconstriction in response to S1P and pressure in normotensive MA.

Figure 2

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Concentration-response curves of ECKO-S1pr1, S1pr3−/− and control MA in response to (A, B) S1P-induced vasoconstriction, n≥7 mice/group (n≥7 MA/group); (Ca, Da) myogenic tone (%) and (Cb, Db) passive tone in response to intraluminal pressure increase, n≥8 mice/group (n≥11 MA/group), and (E, F) PE-mediated vasoconstriction, n≥5 mice/group (n≥9 MA/group). Data were expressed as the mean ± s.e.m. **P<0.01 and ***P<0.001 compared to control mice. Statistical significance was determined by Two-way ANOVA.

S1PR1, but not S1PR3 signaling, maintains low BP in physiological and pathological conditions

Whereas the role of S1PR1 has been elegantly studied in the vasculature during embryogenesis2, its role in BP homeostasis in the adults is largely unknown. ECKO-S1pr1 mice showed higher BP than S1pr1f/f at baseline as well as following AngII chronic infusion (Fig. 3A), underlying anti-hypertensive functions of S1PR1 signaling. Ach-induced vasodilation was impaired to the same extent in hypertensive and normotensive ECKO-S1pr1 and S1pr1f/f MA, indicating the same degree of endothelial dysfunction (Fig. 3B). Interestingly, the vasorelaxation to flow was already blunted in ECKO-S1pr1 MA at baseline and was no further affected by Ang-II treatment (Fig. 3Ca), suggesting that S1PR1 specifically mediates the vasodilation in response to flow upstream the NO pathway. At baseline, ECKO-S1pr1 MA vascular tone was augmented at baseline (Fig. 3Cb), whereas PE-induced vasoconstriction was not affected (Fig. 3Da,b).

Figure 3. S1PR1 signaling exerts anti-hypertensive and vasculo-protective functions.

Figure 3

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(A) SBP was measured with tail-cuff system in S1pr1f/f and ECKO-S1pr1 mice before and after AngII-osmotic pump implantation twice/week for 28 days. ECKO-S1pr1 and S1pr1f/f MA were assessed for vascular reactivity in response to (B) Ach, n=5 mice/group (n≥8 MA/group), (Ca) changes in flow, n=5 mice/group (n≥6 MA/group); (Cb) myogenic tone (%), n=5 mice/group (n≥7 MA/group) and (Da, b) PE, n=5 mice/group (n≥6 MA/group). (Ea) Immunoblot analysis for S1PR1 in thoracic aortas of normotensive and hypertensive WT mice and (Eb) relative quantification of S1PR1 expression to β-actin (n=5 mouse aorta/group). (Ec) S1P-induced vasodilation in normotensive and hypertensive WT MA, n=4 mice/group (n=4 MA/group). (F) Immunofluorescent staining for S1PR1 (red) and isolectin (IB4, green) in normotensive and hypertensive WT, ECKO-S1pr1 MA. Nuclei are stained with DAPI (blue). Data were expressed as mean ± s.e.m. **P<0.01 and ***P<0.001 compared to S1pr1f/f unless otherwise indicated; # #P<0.01 and # # #P<0.001 hypertensive S1pr1f/f vs. normotensive S1pr1f/f; $$$P<0.001 hypertensive ECKO-S1pr1 vs. normotensive ECKO-S1pr1. Statistical significance was determined by One- or Two-way ANOVA.

Next, we assessed S1PR1 levels in hypertension. Western blot analysis of aortas and relative quantification (Fig. 3Ea,b) and immunofluorescent staining of MA (Fig. 3Ec) showed that S1PR1 expression was not altered in hypertension. However, S1P-induced vasodialtion was significantly reduced in MA from Ang-II-treated mice (Fig. 3F), most likely because the impairment in the downstream NO production during hypertension23.

Importantly, the loss of S1PR3 did not alter the onset of hypertension (Fig. S2 A), as well as the vasodilation in response to Ach, flow and S1P in hypertensive MA vs. WT (Fig. S2 Ba–c). On the contrary, vasoconstriction to PE, pressure and S1P were markedly reduced (Fig. S2 Ca–c), indicating a prevailing role of S1PR3 in the VSMC vs. EC functions.

Collectively, these data demonstrate that S1PR1 signaling is a key pathway in BP homeostasis and when genetically ablated, BP is re-set to a higher-then-normal values in both physiological and pathological conditions.

Chronic administration of FTY720 increases BP in normotensive mice and exacerbates hypertension induced by chronic infusion of Ang-II

The anti-hypertensive effects of SEW28716 and the increase in SBP following genetic depletion of S1pr1 in the endothelium raises questions of the effects of FTY720 on the CV system, especially considering that when given chronically FTY720 acts as functional antagonist of S1PR124, 25. In patients, FTY720 induces bradycardia within six hours following the first dose17, 26 and a slight increase of BP (about 2–5 mmHg) with chronic treatment17, 20. In rodents, the administration of FTY720 for a period of ten days significantly raised the BP in rats in a dose-dependent manner27, while a single dose of FTY720 further increased the BP of hypertensive SHR rats at 24h post-administration28, suggesting that drugs inhibiting S1PR1 signaling may be deleterious in hypertension.

Thus, we sought to investigate in details, the effect of FTY720 on vascular tone and BP regulation in normotensive and hypertensive mice. Chronic administration of FTY720 (0.3 mg/Kg, every other day for five weeks) in C57BL/6 transiently increased SBP at day 7, and constantly raised SBP from day 10 up to ~130 mmHg (Fig. 4A). The levels of S1PR1 in MA from mice treated with FTY720 were undetectable in the endothelium and in the adventitia compared to the vehicle-treated group (Fig. 4B).

Figure 4. Chronic administration of FTY720 0.3 mg/Kg increases SBP in normotensive mice and induces vascular dysfunction.

Figure 4

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Male C57/Bl6 mice were treated with FTY720 0.3mg/Kg or vehicle p.o. for 35 days and (A) SBP was measured with tail-cuff system twice/week, n=5 mice/group. (B) Immunofluorescent staining for S1PR1 (red) and isolectin (IB4, green) in MA isolated from FTY720 (0.3mg/Kg) or vehicle-treated WT mice. Nuclei were stained with DAPI (blue). MA from FTY720 0.3mg/Kg or vehicle treated-group were assessed for vascular reactivity in response to: (Ca) Ach, n≥5 mice/group (n≥10 MA/group); (Cb) S1P, n≥5 mice/group, (n≥5 MA/group); (Cc) stepwise-increase in flow, n≥5 mice/group (n≥9 MA/group). (Cd) NOx plasma levels were quantified with Griess reaction method. n≥5 mice/group. Vasoconstriction in response to (Da) PE, n≥5 mice/group, (n≥10 MA/group); (Db) S1P, n≥5 mice/group, (n≥5 MA/group); (Dc) increase in intraluminal pressure and expressed as percent of myogenic tone calculated as described in Methods, n≥5 mice/group (n≥7 MA/group). (Dd) Endothelium-independent vasodilation to Sodium Nitroprussiate (SNP, an NO-donor), n≥4 mice/group, (n≥4 MA/group). Data were expressed as the mean ± s.e.m. *P<0.05, **P<0.01 and ***P<0.001 compared to vehicle-treated group. Statistical significance was determined by Two-way ANOVA or unpaired T-test.

The endothelial-dependent vasodilation in response to Ach, S1P and flow was markedly reduced in MA from FTY720-treated mice (Fig. 4Ba–c) as well as plasma levels of NOx (Fig. 4Bd). Whereas the vasoconstriction induced by PE and pressure was not different, S1P-induced contraction was significantly increased (Fig. 4Ca–c), most likely because of the downregulation of endothelial S1PR1. Surprisingly the vasodilation induced by SNP, a NO donor, was also impaired (Fig. 4Cd), suggesting that long-term FTY720 treatment impairs the endothelial-dependent and independent vasodilation.

Next, we assessed the therapeutic effects of FTY720 treatment in hypertension. Chronic administration of FTY720 (0.30 mg/Kg) in Ang-II-infused mice induced a significant and prolonged increase in SBP compared to vehicle-treated mice (Fig. 5A). The expression of S1PR1 was not detectable by immunofluorescence in MA from FTY720-treated mice (Fig. 5B). Functionally, FTY720-treatment markedly reduced S1P-induced vasodilation, corroborating the functional antagonism of FTY720 on S1PR1 (Fig. 5C). Ach-mediated vasodilation of hypertensive MA was significantly reduced by FTY720 (Fig. 5D), whereas no differences were observed in response to flow (Fig. 5C), most likely because it was already suppressed in vehicle-treated MA. Furthermore, FTY720 did not alter VSMC contractility in response to PE, S1P as well as intraluminal pressure (Fig. S3), as well as MA morphometry (Fig. S3Cb).

Figure 5. FTY720 0.3 mg/Kg exacerbates the hypertension in AngII-treated mice and impairs the response to S1P.

Figure 5

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C57BL/6 mice infused with Ang-II were chronically treated with FTY720 (0.3 mg/Kg) or vehicle p.o. and (A) SBP monitored by using tail-cuff system, n=6 mice/group. (B) Immunofluorescent staining for S1PR1 (red) and isolectin (IB4, green) of MA isolated from hypertensive WT mice treated with FTY720 (0.3mg/Kg) or vehicle. Nuclei (blue) were stained with DAPI. MA from both groups were assessed for vascular reactivity in response to (C) S1P, n=6 mice/group (n≥6 MA/group); (D) Ach, n=6 mice/group, (n≥6 MA/group); (E) stepwise increase in flow, n=6 mice/group (n≥6 MA/group). (F) NOx plasma levels were quantified with Griess reaction method. n≥7 mice/group. Data were expressed as the mean ± s.e.m. *P<0.05 and ***P<0.001 compared to vehicle-treated group or unless otherwise indicated. Statistical significance was determined by One- or Two-way ANOVA.

At a lower dose, FTY720 (0.15mg/Kg) did not alter the onset of hypertension (Fig. S4A). Whereas S1P-mediated vasodilation was significantly reduced, the response to Ach of MA was improved (Fig. S4Ba,b). However, no differences were observed in response to changes in flow (Fig. S4Bc), pressure, PE and S1P (Fig. S4Ca–c).

Altogether, these findings suggest that long-term treatment of FTY720 at 0.3 mg/Kg exacerbates Ang-II-induced hypertension in vivo, in part through the downregulation of endothelial S1PR1 signaling, underlining the importance of this pathway in maintaining vascular homeostasis.

DISCUSSION

Hypertension, the leading cause for myocardial infarction, heart failure and stroke29, is multifactorial and highly complex. Despite current therapies, BP management does not reach a satisfactory outcome in all the patients, suggesting the need for alternative therapeutic targets and a better understanding of underlying mechanisms. The results of this study reveal S1PR1 signaling as a necessary pathway to preserve BP homeostasis. Indeed genetic and pharmacological (FTY720) depletion of S1PR1 impairs vascular functions and increases BP. Furthermore, our data identify a novel role of S1PR1 in mediating the response of resistance arteries to flow, thus acting as mechanoreceptor.

Recently, we reported that endothelial derived S1P has a great impact on systemic BP regulation via S1PR1-S1P-eNOS autocrine signaling6, 11, 22. Shear stress exerted by flowing blood induces the production and release of S1P by the endothelium30, contributing to the physiological activation of S1PR1 signaling in the vasculature6. Pharmacological inhibition of S1PR1 in MA significantly reduced flow-mediated vasodilation6, 11, and lack of S1PR1 in EC in vitro and thoracic aorta in vivo reduced eNOS activation in response to shear stress22, implicating S1PR1 signaling in the flow-mediated vasodilation upstream of NO production.

Shear stress induces the release of vasodilatory factors, of which NO is the predominant mediator of the vasorelaxation induced by flow31, 32. Despite the broad effort to identify the cellular mechanisms of mechanotransduction, how mechanical forces are translated in biological and biochemical responses remain unclear. Multiple mechanotransduction signaling have been suggested, including ion channels33, 34, Pecam-135, the complex Pecam-1/VE-cadherin/VEGFR236, primary cilium37 and glycocalyx38. A recent study of Wang S. et al. demonstrated an important role for P2Y2 receptor coupled to G proteins Gq and G11 in flow-induced activation of eNOS, hence vascular tone and BP regulation in vivo39. Under flow conditions, EC release ATP40, which in turn, can bind to P2Y2 receptors in autocrine/paracrine manner and trigger the downstream activation of AKT-eNOS signaling39. In addition to ATP, shear stress also stimulates endothelial release of S1P30. S1PR1 was shown to be necessary for flow-mediated primary vascular network formation in the retina, alignment of endothelial cells in response to flow-mediated Akt and eNOS activation22, and vasodilation of resistance arteries in response to flow6, 11, suggesting its critical involvement in biomechanical signaling in the endothelium.

Here, we demonstrated that the loss endothelial S1PR1 or FTY720 treatment abolished flow-dependent vasorelaxation, reduced plasmatic levels of NOx and reset system BP to a higher-than-normal value, providing compelling and direct evidence of the importance of S1PR1 as critical mechanosensing pathway in BP homeostasis. Additionally, endothelial S1R1 also preserves vascular structure in adult mice, as ECKO-S1pr1 MA showed an outward remodeling. This is not surprising since global S1pr1-KO mice are embryonically lethal due to a defect in smooth muscle cell coverage and vascular maturation1.

Initial studies demonstrated that vasoconstriction of basilar arteries in response to S1P is mainly mediated by S1PR3 and not S1PR29, 10. However, the lack of S1PR2 does not alter systemic BP41, indicating suggesting a minor role of this receptor. A recent study from Hoefer et al. reported S1PR2 as key regulator of myogenic tone in MA during heart failure conditions42. Our data demonstrated an important and specific role of S1PR3 in vascular tone induced by S1P and pressure, although its loss did not affect the systemic BP in normotensive and hypertensive conditions. BP is not affected by the deletion of either S1pr241 or S1pr3, possibly due to a compensatory mechanism by either one of these receptors.

The loss of S1PR3 did not affect the vasorelaxation induced by low concentrations of S1P. These data suggest a prevailing function of VSMC vs. EC S1PR3 in vascular tone regulation. This finding is not in contradiction with the study published few years ago by Noefer et al. demonstrating that S1P- and HDL-induced vasodilation was reduced in the thoracic aorta rings from S1pr3−/− mice13. In this study the authors used capacitance artery (thoracic aorta), whereas here we employed resistance arteries MA from S1pr3−/− mice, more relevant to BP regulation. It is likely that endothelial S1PR3 plays an important role in the HDL-bound S1P mediated atheroprotective functions in capacitance arteries, whereas in resistance arteries the smooth muscle S1PR3 controls vascular tone.

Previous studies reported that locally produced S1P is important in myogenic tone regulation42–44, in part via S1PR2 activation42. Here, we unveiled an important role also for S1PR3 in myogenic tone regulation.

Lastly, FTY720 treatment markedly raises BP in normotensive and hypertensive mice also underlie the importance of S1P signaling in maintaining BP homeostasis. Whereas it is difficult to tease out mechanisms accountable for the FTY720-induced elevation in BP in vivo, in ex-vivo studies of resistance arteries FTY720 reduced the vasodilation induced by S1P and Ach in the endothelium as well as VSMC relaxation in response to SNP, a NO donor. Our data suggest that long-term administration of FTY720 impairs not only the endothelial, but also smooth muscle cells functions by impairing the NO-induced activation of guanylyl cyclase and cGMP-mediated vasorelaxation. S1P vasodilation is imputable to both, downregulation of S1PR1 by FTY720 and NO-cGMP signaling. Our data are also in agreement with a previous study from Spijkers et al. showing an increase in BP of spontaneously hypertensive rats when treated with one dose of FTY72028 and the recent study of Fryer et al. demonstrating a dose-dependent increase in BP in rats following chronic FTY720 administration27.

The finding that S1PR1 signaling plays an important role in the mechanotransduction of the endothelial shear stress to impact vascular tone and BP homeostasis is novel and of great significance for better understand the molecular mechanisms of the endothelial dysfunction not only in the pathogenesis of hypertension but also in other cardiovascular diseases where endothelial dysfunction is a common denominator.

PERSPECTIVES

Although our knowledge on S1P has substantially increased in the recent years, the regulation and function of S1P signaling in BP homeostasis, and in other cardiovascular diseases, remains largely unknown. Our study reveals an important role of S1P signaling in the pathogenesis of hypertension, and establishes the foundation for therapeutic intervention on the S1PR1 signaling pathway. A better understanding of the role of S1P signaling in cardiovascular diseases has the potential to be rapidly translated, especially considering that this pathway is already therapeutically targeted in autoimmune diseases.

Supplementary Material

Material and Methods

Figure 6. Endothelial S1PR1 is a key regulator of blood flow and pressure.

Figure 6

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In addition to mediate S1P-induced vasodilation, this study revealed an important role of endothelial S1PR1 in systemic BP regulation and biomechanical signaling, and unveils potential cardiovascular side effects of FTY720, S1PR1 functional antagonist.

The increase in flow induces the release of S1P by the endothelium, which activates S1PR1 through an autocrine/paracrine manner leading to eNOS-derive NO production and vasodilation. On the contrary, S1PR3 signaling plays a prevalent role in regulating the tone of the smooth muscle layer in response to S1P and intraluminal pressure increase but has no effect on SBP. Chronic administration of FTY720 downregulates S1PR1 in the endothelium and adventitia of the MA and markedly impairs vascular functions leading to systemic increase of BP.

NOVELTY AND SIGNIFICANCE.

What Is New?

  • In this study we discovered a novel and important role of S1PR1 signaling in preserving BP homeostasis in physiological and pathological conditions.

  • Mechanistically, S1PR1 functions as mechanotransduction signaling in response to flow and maintains vascular structure in mesenteric arteries.

  • S1PR3 in VSMC controls MA contractility in response to pressure and S1P, whereas S1PR3 in the endothelium is dispensable in vascular tone regulation.

  • FTY720 downregulates endothelial S1PR1, increases BP of normotensive mice and exacerbates angiotensin-II-induced hypertension in a dose-dependent manner.

What Is Relevant?

  • The identification of a novel therapeutic pathway for the treatment of hypertension.

  • Potential cardiovascular side effects in patients chronically treated with FTY720

Summary

  • S1P-S1PR1 signaling is a novel mechanotransduction signaling in the endothelium mediating the vasodilation to flow and lowering BP.

Acknowledgments

SOURCE OF FUNDING

This work was supported by NIH grant R01HL126913 to A. Di Lorenzo and R01HL89933 and Fondation Leducq trans-atlantic network grant to T. Hla.

Nonstandard Abbreviations and Acronyms

S1P

S1P

S1PR1

S1P receptor 1

S1PR3

S1P receptor 3

eNOS

endothelial nitric oxide synthase

NO

nitric oxide

VSMC

vascular smooth muscle cells

MA

mesenteric artery

SBP

systolic blood pressure

HDL

high-density lipoprotein

WT

wild type

Footnotes

DISCLOSURE

None

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Material and Methods
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