Quick search in Citations
This JournalAnywhereCitation
Advanced Search
Research Article
Originally Published 2 December 2020
Free Access

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke

Anja Nitzsche https://orcid.org/0000-0003-0567-6790, Marine Poittevin, Ammar Benarab, Philippe Bonnin https://orcid.org/0000-0003-3184-3740, Giuseppe Faraco https://orcid.org/0000-0001-8675-4580, Hiroki Uchida, Julie Favre, … Show All … , Lidia Garcia-Bonilla, Manuela C.L. Garcia, Pierre-Louis Léger, Patrice Thérond, Thomas Mathivet https://orcid.org/0000-0001-7761-1684, Gwennhael Autret https://orcid.org/0000-0001-7335-350X, Véronique Baudrie, Ludovic Couty, Mari Kono https://orcid.org/0000-0003-2447-4350, Aline Chevallier, Hira Niazi https://orcid.org/0000-0003-4737-2613, Pierre-Louis Tharaux https://orcid.org/0000-0002-6062-5905, Jerold Chun, Susan R. Schwab, Anne Eichmann, Bertrand Tavitian https://orcid.org/0000-0002-5349-8194, Richard L. Proia, Christiane Charriaut-Marlangue https://orcid.org/0000-0002-9632-5453, Teresa Sanchez https://orcid.org/0000-0002-2711-7423, Nathalie Kubis, Daniel Henrion https://orcid.org/0000-0003-1094-0285, Costantino Iadecola https://orcid.org/0000-0001-9797-073X, Timothy Hla https://orcid.org/0000-0001-8355-4065, and Eric Camerer https://orcid.org/0000-0002-6271-7125 [email protected]Author Info & Affiliations
Circulation Research
Volume 128, Number 3
https://doi.org/10.1161/CIRCRESAHA.120.316711
Track CitationsAdd to favorites
PDF/EPUB
VIEW EDITORIAL:New Vistas in Stroke Treatment

Graphical Abstract

Abstract

Rationale:

Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke.

Objective:

To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy.

Methods and Results:

Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion.

Conclusions:

This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Introduction

Editorial, see p 383
Meet the First Author, see p 307
Ischemic stroke is one of the most prevalent causes of death and morbidity worldwide and represents a major societal and economic burden (https://www.who.int).1 Despite several clinical trials with neuroprotective therapeutics, treatment options remain limited to thrombolysis and mechanical recanalization.2 Novel safe and affordable adjunct treatment strategies are, therefore, needed.
Ischemic stroke is caused by thrombotic or embolic occlusion of a large cerebral artery. This produces a core of necrotic tissue immediately downstream of the occlusion site surrounded by an ischemic penumbra that can be rescued if adequate perfusion is sustained.1,3 The speed by which the core encroaches upon the penumbra depends on the efficiency of retrograde blood supply through cortical collateral anastomoses extended across the borders with neighboring cerebral arterial territories. Although governed primarily by the number and size of existing anastomoses, blood rerouting also depends on the dilatory capacity, integrity, and patency of the regional vasculature.4 Improving microvascular function to counteract the expansion of the infarct core and improve the efficacy and safety of anterograde reperfusion may, therefore, provide a therapeutic opportunity.5 Strategies explored to this end include the inhibition of lymphocyte-driven inflammatory thrombosis and the stimulation of endothelial cell (EC) function to promote local redistribution of blood flow, reinforce the blood-brain barrier (BBB), and suppress inflammation and coagulation in affected territories.5,6
S1P is a signaling lipid with critical roles in both immune and vascular function exerted by 5 cognate GPCRs (G protein-coupled receptors), S1P1-5.7 Lymphocytes depend on S1P1-mediated S1P sensing to egress from lymphoid organs, and both inhibition and activation of lymphocyte S1P1 induce profound lymphopenia.8 S1P1 is also among the most abundant EC GPCRs, and selective constitutive or temporal deletion of S1pr1 (encoding S1P1) in mouse ECs impairs embryonic and postnatal angiogenesis, vascular integrity, and flow-mediated vasodilation.7,9,10 Loss of S1P1 signaling in ECs destabilizes adherens junctions, reduces eNOS (endothelial nitric oxide synthase) activity, and increases the expression of leukocyte adhesion molecules.10–12 S1P1 thus plays a critical role in sustaining hallmark endothelial functions. S1P is abundant in circulation, where it associates primarily with HDL (high-density lipoprotein) and albumin.7 Erythrocytes and ECs are the main sources of blood and lymph S1P under homeostasis, whereas platelets store large amounts of S1P that is only released upon activation.7,9,13 S1P is exported from ECs by Spns2 (spinster homolog 2) and from platelets and erythrocytes by Mfsd2b (major facilitator superfamily domain containing 2B).7,14
The multiple sclerosis drug fingolimod (FTY720) is a functional antagonist of S1P1 that also activates S1P3,4,5 and has shown promise for the treatment of ischemic and hemorrhagic stroke in both experimental models and small-scale clinical trials.15–24 Similar efficacy of S1P1-selective agonists argues that FTY720 protection is mediated by S1P1.6,19,21 Loss of efficacy in lymphocyte-deficient mice and correlation between efficacy on lymphocyte depletion and infarct reduction has indicated that S1P1 modulators provide protection by impairing lymphocyte trafficking and, thereby, lymphocyte-driven thromboinflammation.19,20 Yet the endothelial receptor pool is also engaged after systemic drug treatment, and the risks and benefits of targeting EC S1P1 have not been specifically evaluated.
In this study, we have addressed the impact of tissue-specific deficiency of S1P1 and sources of its activating ligand on the development, anatomy, and function of the naive brain vasculature and on the outcome of transient and permanent middle cerebral artery occlusion (tMCAO and pMCAO, respectively) in mice. This revealed an important role for EC S1P1 in cerebrovascular homeostasis and a critical protective role for EC-autonomous engagement of S1P1 signaling during cerebral ischemia. When we addressed the capacity of pharmacological agonists to access S1P1 on the brain endothelium, we found that BBB penetration is required for engagement of EC S1P1 and that this receptor pool is an important target for the protective effects of S1P receptor modulation in ischemic stroke.

Methods

Detailed Methods are available in the Data Supplement.

Data Availability

The authors declare that all supporting data are available within the article and its Data Supplement.

Results

Endothelial S1P1 Plays a Critical Protective Role During Cerebral Ischemia

To define endogenous roles of EC and leukocyte S1P1 in ischemic stroke, we generated EC S1pr1 knockout mice with Cdh5-iCreERT2 or Pdgfb-iCreERT2 (S1pr1ECKO) and pan-hematopoietic S1pr1 knockout mice with Mx1-Cre or Vav1-Cre (S1pr1HCKO).9,11,25 The Pdgfb-iCreERT2 allele induced near-complete recombination of an mTmG (membrane targeted tandem dimer Tomato/membrane targeted green fluorescent protein) reporter in ECs in the cerebral cortex after neonatal tamoxifen administration (Figure IA in the Data Supplement). We have previously reported efficient S1pr1 excision in target tissues with the other Cre drivers.25,26 In a pMCAO model, in which thermocoagulation and subsequent dissection of a section of the MCA distal to the lenticulostriate arteries yields small and cortically confined infarcts but no overt neurological deficits,27,28 mean 24-hour infarct volumes were on average 71% larger in neonatally induced S1pr1ECKO males and 65% larger in S1pr1ECKO females than in littermate controls (Figure 1A). The relative increase was greater 3 days after pMCAO with 148% larger infarcts in S1pr1ECKO males (Figure 1B). Although the Pdgfb-iCreERT2 allele used for EC-selective gene deletion has off-target effects in megakaryocytes,25,29 the phenotype could be attributed to ECs as it was reproduced in S1pr1ECKO males generated with the EC-selective Cdh5-iCreERT2 (Figure 1C), but not with S1pr1MKKO males generated with the megakaryocyte-selective Pf4-Cre (Figure IB in the Data Supplement).25 In the same experimental model, hematopoietic S1P1 deficiency induced profound lymphopenia but had no impact on infarct size (Figure 1D, Figure IC in the Data Supplement). Similar results were obtained in a filament-based proximal transient MCAO model, in which infarct volumes measured 24 hours after 60 minutes occlusion were on average more than twice as large and neurological deficits exacerbated in S1pr1ECKO mice (Figure 1E). Induction of gene deletion 10 days before surgery in this experiment argued that sensitization to MCAO did not reflect upon developmental consequences of EC S1pr1 deletion. Yet as control infarcts were smaller than typically observed with proximal MCAO—an observation that may be attributed to protective effects of tamoxifen30,31—neonatal deletion was used in all subsequent experiments. When neonatally induced S1pr1ECKO males were subjected to a severe tMCAO model in which reperfusion was delayed to 90 minutes, infarcts covered a substantial portion of the MCA territory in controls, and S1pr1ECKO mice exhibited considerable postreperfusion mortality (68% versus 32%, respectively, P=0.022; Figure ID in the Data Supplement) that manifested >8 hours after occlusion. Postmortem analysis did not reveal bleeding, and hemorrhagic transformation was not increased in S1pr1ECKO survivors (minor bleeds were observed in 1/6 S1pr1ECKO versus 5/14 control infarcts). Loss of the most affected animals may explain a lack of significant difference in infarct volumes observed in survivors (Figure ID in the Data Supplement) and is further addressed below. Unlike the pMCAO model, hematopoietic S1P1 deficiency afforded some neuroprotection 24 hours after 60 minutes tMCAO (Figure 1F; P=0.074). These observations reveal a critical role for endothelial S1P1 in limiting neuronal injury in ischemic stroke irrespective of whether or not the occluded artery is reperfused (Figure 1G). They also support the notion that S1P1 plays a disruptive role in ischemic stroke by supporting lymphocyte egress,20 yet the effect of specifically impairing this pathway was modest and observed only in the context of reperfusion (Figure 1G).
Figure 1. Endothelial S1P1 (sphingosine 1-phosphate receptor-1) signaling limits brain injury after permanent and transient middle cerebral artery occlusion (pMCAO and tMCAO, respectively). A–C, Infarct volumes 24 (A and C) or 72 (B) hours after pMCAO in S1pr1ECKO and littermate males (A, middle, B) and females (A, right) generated by neonatal S1pr1 deletion with Pdgfb-iCreERT2 (A and B) or Cdh5-iCreERT2 (C). Left of A, Representative images. D, Basal peripheral blood lymphocyte counts and infarct volumes 24 h after pMCAO in male mice lacking S1P1 in hematopoietic cells (S1pr1HCKO; Vav1-Cre). E, Infarct volumes and neurological deficits 24 h after 60 min tMCAO in male mice lacking S1P1 in endothelial cells (S1pr1ECKO; Cdh5-iCreERT2; adult deletion). Left, Representative images. F, Infarct volumes 24 h after 60 min tMCAO in males lacking S1P1 in hematopoietic cells (S1pr1HCKO; Mx1-Cre). Lymphocyte counts pre-MCAO in right. G, Schematic representation of the net cell type-specific contribution of S1P1 signaling to stroke outcome. Bar graphs show mean±SEM. Statistical significance assessed by Mann-Whitney test (A, males) or unpaired t test (all other).

Ischemia Mobilizes EC-Autonomous S1P Provision for S1P1 Signaling and Stroke Protection

S1P1 drives lymphocyte egress and sustains lung vascular integrity in response to circulating ligand.13,32 Surprisingly, however, postnatal deletion of Sphk1&2 in Mx1-Cre sensitive cells did not significantly impact infarct volumes after pMCAO or tMCAO (Figure 2A), even if the same deletion strategy nearly abolishes S1P provision to plasma, resulting in lymphopenia and constitutive vascular leak in the lung.9,13,32 This could reflect compound effects of loss of S1P production also in tissue-resident cells or of loss of circulating S1P on the activation of S1P1 and other S1PRs, notably S1P2,33 in several cellular compartments.34 However, lymphocyte S1P1 was dispensable in the pMCAO model (Figure 1D), and deletion of S1pr2 or S1pr3 did not impact outcome in the pMCAO model (Figure IIA in the Data Supplement). Selective impairment of S1P release from platelets, which could potentially increase local S1P levels in stroke and change signaling bias,9,35 did not impact infarct size in the tMCAO model (Figure 2B). Single-cell RNA sequencing analysis suggests that brain ECs express the necessary machinery for de novo S1P synthesis and export and may thus constitute a local source of S1P in the neurovascular unit.36 Accordingly, impairment of the production (Sphk1&2ECKO) or the export (Spns2ECKO) of S1P in ECs both exacerbated outcome to a similar degree as EC S1P1 deficiency in the pMCAO and the tMCAO model, pointing to a critical role for cell-autonomous S1P provision for EC S1P1 activation (Figure 2C and 2D and Figure IIB and IIC in the Data Supplement). Excision efficiency was confirmed to be >85% for Sphk1 and Sphk2 in isolated brain ECs from Sphk1&2ECKO (Figure IID in the Data Supplement). To address where cell-autonomous S1P1 signaling is engaged after MCAO, we first asked where S1P1 is expressed. Single-cell RNA sequencing of brain microvascular fragments shows enrichment of S1pr1 transcripts in ECs as well as in astrocytes (Figure IIE in the Data Supplement).36 Accordingly, protein expression, which initially appeared widespread and diffuse in the cerebral cortex (Figure 2E), could be resolved by selective deletion of S1pr1 in ECs (S1pr1ECKO) or astrocytes (S1pr1ACKO; Figure 2E, Figure IIF and IIG in the Data Supplement). The level of EC S1P1 expression is maintained in old mice (Figure IIG and IIH in the Data Supplement).37 We then generated S1P1GS (S1P1 green fluorescent protein [GFP] signaling) mice—which leave a nuclear GFP signal after S1P1-β-arrestin coupling38—with or without the capacity for EC S1P production (S1pr1Ki/+:H2B-GfpTg/+:Sphk1&2ECWT/KO). Despite widespread receptor expression in astrocytes and ECs throughout the vascular tree, S1P1 signaling was highly restricted to a subset of arteriolar ECs in both young and old mice (Figure 2F, Figure III and IIJ in the Data Supplement). S1P1-independent H2B-GFP activity was also observed in perivascular cells of arterioles and venules but not in ECs (Figure IIK in the Data Supplement). After pMCAO, S1P1 signaling in the infarct region expanded to capillary and venous ECs, but not to astrocytes (Figure 2G and 2H). While redundant ligand sources9 or ligand-independent activation11 sustained homeostatic signaling in arterioles (Figure 2H), the expansion of S1P1 signaling after MCAO was driven principally by EC-autonomous S1P production (Figure 2H). To address the mechanistic basis for this expansion, we evaluated the expression of Sphks, Spns2, and S1P1 in brain microvessels 6 hours after tMCAO. Consistent with single-cell RNA sequencing data,36 Sphk2, Spns2, and S1pr1 and to a lesser degree Sphk1 were all expressed in cerebral microvessels of naive mice. Ischemia triggered robust induction of Sphk1 expression in the ipsilateral hemisphere but had little impact on the expression of the other transcripts (Figure 2I, Figure IIL in the Data Supplement). Thus, while circulating S1P sustains lymphocyte trafficking and may contribute to homeostatic signaling in cerebral arterioles, S1P1-dependent neuroprotection in ischemic stroke is driven primarily by engagement of EC-autonomous S1P provision, possibly by transcriptional activation of Sphk1.
Figure 2. Endothelial cell (EC) autonomous S1P (sphingosine 1-phosphate) provision sustains S1P1 (S1P receptor-1) activation during cerebral ischemia. A–D, Infarct volumes 24 h after permanent middle cerebral artery occlusion (pMCAO) or 60 min transient middle cerebral artery occlusion (tMCAO) in male mice lacking S1P production in hematopoietic cells (Sphk1&2HCKO; A), platelets (Sphk2MKKO; B), or endothelial cells (Sphk1&2ECKO; C), or deficient in S1P export from blood endothelial cells (Spns2ECKO; D) and respective littermate controls. E, S1P1 expression in the naive cerebral cortex of wild-type mice (left) and mice lacking S1P1 in astrocytes (S1pr1ACKO, Gfap-Cre, right). Note expression of S1P1 in all vessels. Green, S1P1; white, ECs (CD31); red, vascular smooth muscle cells (ASMA); blue, all cell nuclei (Hoechst). Scale bar: 50 μm. F and G, S1P1 signaling visualized in S1P1 signaling mice (S1P1GS) in a naive cerebral cortex (F) and 48 h after pMCAO (G) assessed in the contralateral (left) and ipsilateral (right) cerebral cortex. Note that S1P1 signaling is highly restricted to arteries in the naive and contralateral cerebral cortex and more widespread but still predominantly endothelial in the ipsilateral cortex. Red, EC nuclei (Erg); green, S1P1 signaling cells (GFP); yellow, S1P1 signaling ECs (GFP/Erg double-positive nuclei); white, ECs (CD31); blue, vascular smooth muscle cells (ASMA). Scale bar: 100 μm. H, Quantification of GFP positive ECs as a fraction of total arterial and nonarterial ECs in the ipsilateral and contralateral hemisphere of S1P1GS mice with (Sphk1&2ECWT) and without (Sphk1&2ECKO) the capacity for EC S1P production 48 h after pMCAO. I, Relative expression of S1pr1, Sphk1, Sphk2, and Spns2 in cerebral microvessels isolated from naive cerebral cortex or 6 h after tMCAO, normalized to Tjp1 transcript. Bar graphs show mean±SEM. Statistical significance assessed by ANOVA with Tukey multiple comparisons test (H and I), Mann-Whitney test (C, pMCAo), or unpaired t test (all other).

Postnatal Impairment of EC-Autonomous S1P Signaling Does Not Impact Cerebrovascular Anatomy

Although recombination of loxP-flanked alleles in ECs in neonatal mice in most experiments in this study overcame confounding effects of tamoxifen protection, it also introduced potential confounding effects of deregulated vascular development on stroke outcome. As previously reported for Cdh5-iCreERT2-mediated neonatal deletion of S1pr1,11 Pdgfb-iCreERT2-mediated deletion induced vascular hypersprouting and delayed outgrowth of the retinal vasculature (Figure 3A and 3B). However, this phenotype was not replicated with Pdgfb-iCreERT2-mediated deletion of Sphk1&2 (Figure 3A and 3B). This suggests that postnatal angiogenesis, like embryonic angiogenesis,9 is sustained by redundant S1P sources and that abnormal vascular patterning is unlikely to explain sensitivity to MCAO in Sphk1&2ECKO mice. Consistent with prenatal development of the cerebral vasculature, we also did not observe significant differences in the number of collateral connections between the MCA and branches of anterior cerebral artery (ACA) extending laterally from the midline between S1pr1ECKO and littermate controls (Figure 3C and 3D). Vascular density in the cerebral cortex was also unaltered by Pdgfb-iCreERT2-mediated S1pr1 excision, as has previously been reported for Cdh5-iCreERT2-mediated deletion (Figure 3E).26 Thus, the increased impact of MCAO in mice deficient in EC-autonomous S1P1 signaling cannot be explained by underlying differences in vascular anatomy.

Endothelial S1P1 Maintains BBB Function

Both genetic strategies for EC S1pr1 deletion employed in this study result in constitutive vascular leak in the lung that can be replicated by plasma but not by EC S1P deficiency.9,39 Cdh5-iCreERT2-mediated S1pr1 deletion results in subtler and size-selective permeability of the brain endothelium,26 which was also observed with Pdgfb-iCreERT2-mediated S1pr1 deletion (Figure 4A). Endotoxin challenge (10 mg/kg, 8 hours) increased 4 kD dextran accumulation to the same degree in S1pr1ECKO and littermate controls (Figure 4B). Thus, S1P1 deficiency does not critically impair the stability of EC junctions at the BBB. Intriguingly, even if S1pr1ECKO mice do not show increased paracellular permeability to dextrans ≥10 kD,26 naive S1pr1ECKO mice did show increased permeability to Evans Blue/albumin, which crosses the BBB primarily by transcellular transport (Figure 4C).40 Neither basal phenotype was replicated in mice lacking EC S1P production nor was Evans Blue/albumin extravasation affected by lack of plasma S1P (Figure 4A and 4C), again suggesting source redundancy or ligand-independence of homeostatic EC S1P1 signaling at the BBB. Twenty-four hours after pMCAO, Evans Blue/albumin accumulation in the ipsilateral hemisphere exceeded the relative increase in infarct size in S1pr1ECKO mice with a more diffuse and widespread appearance and was also significantly higher in the contralateral hemisphere, consistent with results in naive mice (Figure 4D). In the acute phase after 90 minutes tMCAO—which is associated with high mortality of S1pr1ECKO mice (Figure ID in the Data Supplement)—full T2 weighted magnetic resonance imaging revealed severe edema in S1pr1ECKO mice as early as 2.5 hours after reperfusion with a clear shift in the midline 2 hours later (Figure 4E). Image analysis confirmed significantly larger T2 lesions, demonstrating a clear impact of EC S1P1 deficiency also after 90 minutes of tMCAO despite no significant increase in infarct volumes in the few S1pr1ECKO mice that survived for 24 hours (Figure ID in the Data Supplement). Transtentorial herniation may be followed by cerebellar tonsil herniation and could explain increased mortality in S1pr1ECKO mice in this model (Figure ID in the Data Supplement). Thus, S1P1 preserves BBB integrity in ischemic stroke most likely by restricting vesicular transport, which underlies BBB dysfunction in the acute phase.40

S1P1 Supports Cerebral Vasoreactivity and Promotes Tissue Perfusion After MCAO

We next addressed if EC S1P signaling regulates vessel diameter and thereby the redistribution of blood to the ischemic penumbra through existing cortical collateral anastomoses. Significantly reduced blood flow responses assessed by Doppler ultrasonography in the somatosensory cortex in response to acetylcholine superfusion (Figure 5A) and in the basilar trunk in response to CO2 inhalation (Figure 5B) both argued a critical role for EC S1P1 in cerebral blood flow regulation. Blood flow responses in the somatosensory cortex in response to whisker stimulation were nevertheless unaltered, suggesting normal neurovascular coupling, as was mean arterial pressure observed during these recordings (Figure 5A, Figure IIIA in the Data Supplement). Flow-mediated dilation was also significantly reduced in posterior cerebral artery segments isolated from S1pr1ECKO mice (Figure 5C). A similar phenotype in mesenteric arteries from both S1pr1ECKO and Sphk1&2ECKO mice (Figure IIIB in the Data Supplement) argued that shear forces can engage EC S1P1 through cell-autonomous S1P release. Impairment of vascular reactivity in S1pr1ECKO mice did not, however, impact central blood pressure (assessed by telemetry; Figure 5D) or basal brain perfusion (assessed by arterial spin labeling magnetic resonance imaging; Figure 5E).41 We next monitored mean blood flow velocities in the left and right internal carotid arteries (ICA) and in the basilar trunk before, 50 and 120 minutes after pMCAO in S1pr1ECKO and littermate controls (Figure 5F). No significant genotype-dependent difference was observed in baseline mean blood flow velocities (Figure IIIC in the Data Supplement). Permanent left MCAO decreased mean blood flow velocities in the left ICA to 77% of preocclusion values 50 minutes after occlusion in both S1pr1ECKO and control mice (Figure 5F). Consistent with this decrease reflecting upon the reduction in MCA territory downstream of the left ICA, we observed no significant change in mean blood flow velocities in the right ICA or the basilar trunk (Figure IIIC in the Data Supplement). A subsequent recovery to near 90% of preocclusion values at 120 minutes after occlusion in controls was interpreted to reflect upon a downstream increase in peri-infarct reflow through branches of the MCA originating upstream from the occlusion and through the distal branches of the ACA and posterior cerebral artery (Figure 5F, Figure IIIC in the Data Supplement). This recovery at 120 minutes after occlusion was absent in S1pr1ECKO mice (Figure 5F, Figure IIID in the Data Supplement), and a significant inverse correlation was observed between relative ICA blood flow at 120 minutes and infarct volumes at 24 hours (Figure 5G). To address perfusion directly in the affected cortex after pMCAO, we next visualized red blood cell flux in the area of collateral anastomoses in the leptomeningeal arteries between the MCA and ACA by sidestream dark-field imaging (Figure 5H, Movies I through III in the Data Supplement). Two hours after pMCAO, unidirectional flow towards the MCA territory was observed in all ACA-MCA collaterals, allowing perfusion of the territory normally supplied by branches of the MCA downstream of, but distal to the occlusion site (Movies I and II in the Data Supplement). Blood moving retrograde switched anterograde up other MCA branches when encountering coagulated blood in the ischemic core (Movie III in the Data Supplement). While the same general pattern was observed in mice of both genotypes, microvascular perfusion in MCA territory proximal to the ACA border was significantly reduced in S1pr1 ECKO mice (Figure 5H, Movie II in the Data Supplement). No significant genotype-dependent difference in ICA blood flow reduction at 50 minutes argued that protective effects of S1P1 signaling take time to establish, possibly because of the need to engage EC-autonomous S1P production through Sphk1 induction (Figure 2I). Accordingly, when occlusion time was reduced to 35 minutes in the tMCAO model, EC S1P1 deficiency no longer influenced infarct size (Figure 5I). These observations are consistent with the notion that EC S1P1 limits the expansion of the necrotic core in the acute phase of ischemic stroke by supporting local vasodilation so as to promote retrograde perfusion of affected MCA territories.

Endothelial S1P1 Signaling Maintains Microvascular Patency in the Ischemic Penumbra

In addition to the active redistribution of blood from neighboring vascular territories, perfusion of the ischemic penumbra is highly dependent on microvascular patency within the affected zone. The recruitment of leukocytes to an activated endothelium in the ischemic penumbra may directly impair capillary blood flow and propagate microvascular thrombosis.42,43 S1P1 helps sustain the anti-inflammatory status of the aortic endothelium,12 and we observed a significant increase in ICAM (intercellular adhesion molecule)-1 in brain homogenates and in postcapillary venules in the cerebral cortex of naive S1pr1ECKO mice (Figure 6A, Figure IVA in the Data Supplement). This increase was not observed with selective impairment of S1P production in endothelial or hematopoietic cells (Figure 6A, Figure IVA in the Data Supplement). In the acute phase after pMCAO (2.5 hours), however, an increase in ICAM-1 expression in the ipsilateral hemisphere of control but no S1pr1ECKO mice overcame the genotype-dependent difference (Figure 6B). MPO (myeloperoxidase) levels were increased in S1pr1ECKO mice (Figure 6B), albeit not beyond the increase in infarct size (Figure 1A). We next stained for erythrocytes, neutrophils, platelets, and fibrin(ogen) in sections of brains perfused transcardially with heparinized saline 3 hours after pMCAO. Plasma serotonin was slightly increased at this time independent of genotype, arguing against a significant difference in platelet activation (Figure IVB in the Data Supplement). We nevertheless observed a striking reduction in the penetration of tomato lectin, infused 15 minutes before transcardial perfusion, into the MCA territory superior/distal to the core in S1pr1ECKO mice (Figure 6C, Figure IVC in the Data Supplement). This again points to collateral failure. Platelets and fibrin(ogen) were observed within both perfused and nonperfused capillaries only superior to the core and correlated with more intense staining for PECAM (platelet endothelial cell adhesion molecule)-1/CD31 (Figure 6C, Figure IVD and IVE in the Data Supplement). Fibrin deposition was more widespread and significantly more abundant in S1pr1ECKO mice (Figure 6C, Figure IVF in the Data Supplement). Occasional neutrophils were observed within capillaries on both sides of the infarct core at similar frequency in both genotypes (Figure IVD, IVF, and IVG in the Data Supplement). These observations confirm that EC S1P1 signaling promotes retrograde perfusion of the ischemic penumbra and suggest that it does so in part by maintaining microvascular patency.

Receptor Polarization Restricts S1P1 Signaling and Ligand Access at the Blood-Neural Barrier

The need for EC-autonomous S1P provision to sustain vascular protective S1P1 signaling in the ischemic brain could be explained by depletion of circulating S1P, as observed in myocardial infarction and during systemic inflammation.9,44 However, plasma and platelet S1P levels were unchanged in the early acute phase of stroke with or without reperfusion (Figure 7A). An alternative explanation could be that receptor polarization restricts access of S1P1 to circulating ligand at the BBB. To address this possibility, we first used confocal microscopy to evaluate the subcellular localization of EC S1P1. Imaging across one or 2 nuclei allowed us to distinguish the EC plasma membranes, and S1pr1ACKO mice allowed us to discriminate between the abluminal plasma membrane and astrocyte end-feet in the cerebral cortex. In the developing retina, S1P1 was present primarily on the luminal surface of capillary ECs, where it colocalized with ICAM-2 (Figure 7B, Figure VA in the Data Supplement). By contrast, expression was predominantly abluminal on capillary ECs in the adult retina (Figure 7C, Figure VB in the Data Supplement), while a subset of arteriolar ECs retained luminal expression (Figure 7D, Figure VC in the Data Supplement). S1P1 expression was also predominantly abluminal on capillary ECs in the adult brain (Figure 7E, Figure VD in the Data Supplement). Polarization was retained during ischemia (Figure VE in the Data Supplement).
To confirm our impression that S1P1 is surface expressed but luminally excluded in the majority of ECs in the cerebral cortex, we again made use of the S1P1 signaling reporter. In naive S1P1GS mice, hepatocytes show no nuclear GFP although they express S1P1 (Figure VIA in the Data Supplement).38 Systemic administration of the potent S1P1-selective agonist RP-001 (0.6 mg/kg)38,45 induced robust S1P1 signaling in hepatocytes as well as ECs of skeletal muscle and lung, but not cerebral cortex (Figure 7F, Figure VI in the Data Supplement). In striking contrast, when injected directly into the brain parenchyma, RP-001 (0.06 mg/kg) substantially increased S1P1 signaling in arteries, capillaries, and veins of the cerebral cortex (Figure 7F). Astrocytes remained GFP negative despite S1P1 expression (Figure VII in the Data Supplement),36 suggesting together with mostly punctate staining (Figure 7E) that the receptor is not expressed on the astrocyte surface under homeostasis. Further attesting to polarization and a role for S1P1 in blood flow regulation, we observed a significant EC S1P1-dependent increase in cortical blood flow by laser Doppler when RP-001 was administered directly into the cerebrospinal fluid for paravascular access46 but not systemically (Figure 7G). Surprisingly, induction of S1P1 signaling in brain ECs, although detectable, was also minimal after systemic administration of high dose FTY720 (2×5 mg/kg), despite strong activation of the S1P1 reporter in other organs (Figure 7F, Figure VI in the Data Supplement). It should be noted that although FTY720 is known to cross the BBB and desensitize S1P1 on the brain endothelium, this has been demonstrated with high doses over extended time.26,47 CYM-5442 is an S1P1-selective agonist reported to distribute rapidly and preferentially to the brain after systemic injection.48 Accordingly, at a dose required to induce S1P1 signaling equivalent to RP-001 (0.6 mg/kg) and FTY720 (2×5 mg/kg) in hepatocytes, CYM-5442 (10 mg/kg) induced signaling also in brain ECs after systemic administration (Figure 7H, Figure VIA in the Data Supplement).
Collectively, these observations argue that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands in capillary, venous, and most arterial ECs once the blood-neural barrier is established, and that BBB penetration is required for agonists to harness this receptor pool. Gradual receptor polarization in all but a small subset of arterial ECs with maturation of the blood-neural barrier provides potential explanation for why cell-autonomous S1P provision is required for broader endothelial S1P1 activation during cerebral ischemia (Figure 2A, 2C, 2D, and 2H), although it is dispensable both for S1P1 activation in the developing retina (Figure 3A) and for homeostatic signaling in cerebral arterioles (Figure 2F and 2H).

A BBB-Penetrating S1P1-Selective Agonist Limits Cortical Infarct Expansion After Both pMCAO and tMCAO

Our results so far argue that optimal therapeutic S1P1 targeting for stroke would transiently suppress lymphocyte trafficking and activate but not desensitize EC S1P1, and that CYM-5442 may be better suited than RP-001 and FTY720. CYM-5442 distributes preferential to brain and has a relatively short plasma half-life (3 hours).48 Accordingly, CYM-5442 induced lymphopenia 3 hours after administration that was evident at 1 mg/kg and as profound as RP-001 (0.6 mg/kg) and FTY720 (1 mg/kg) at 3 mg/kg (Figure 8A). Twenty-four hours later, lymphocyte counts remained low in mice receiving FTY720 but returned to normal in mice receiving CYM-5442 and RP-001 (Figure 8A). In the pMCAO model, CYM-5442 modestly reduced 24-hour infarct volumes at 1 mg/kg (Figure VIIIA in the Data Supplement) and provided substantial benefit when administered both immediately and up to 6 hours after occlusion at 3 mg/kg (Figure 8B). In a modified pMCAO model that included permanent ligation of the ipsilateral CCA, infarct volumes were also reduced at 7 days with daily CYM-5442 (3 mg/kg) administration (Figure VIIIB in the Data Supplement). Consistent with the engagement of and dependence on EC S1P1, CYM-5442 (3 mg/kg) reversed sensitivity to pMCAO in Sphk1&2ECKO mice (Figure 8C versus Figure IIB in the Data Supplement), but not in S1pr1ECKO mice (Figure 8D versus 1A). CYM-5442 (3 mg/kg) also afforded significant protection when administered during reperfusion 60 minutes after MCAO (Figure 8E). Consistent with a mechanism involving collateral blood supply,28 protection in tMCAO was delimited to the cortex, where an infarct reduction of 70% mirrored protection achieved in the cortically restricted pMCAO model (Figure 8E). RP-001, which did not efficiently cross the BBB (Figure 7F), did not provide protection in the pMCAO model despite inducing equivalent lymphopenia and strong S1P1 signaling in other organs (Figure 8A and 8F, Figure VIA in the Data Supplement). Thus, optimal S1P1 targeting for ischemic stroke requires BBB penetration for engagement of endothelial receptors (Figure 8G) and can provide substantial protection against cortical infarct expansion independent of reperfusion and in a therapeutically relevant time window.

Discussion

S1P1 modulators have shown promise in experimental models and small-scale clinical trials of ischemic and hemorrhagic stroke.6,22 Yet as their mechanisms of action are not fully elucidated, optimal drug properties and targeting strategies remain to be defined. As protection has been attributed to suppression of lymphocyte-mediated thromboinflammation, most current strategies focus on inhibiting the function of lymphocyte S1P1.6,19,20,24 Using a variety of genetic and experimental murine models to address endogenous roles of S1P1 signaling in cerebral ischemia, we confirm the potential benefit of targeting lymphocyte receptors but also reveal a critical role for EC S1P1 in cerebrovascular homeostasis and endogenous protection against ischemic brain damage. Compromised BBB function, reduced vasodilatory capacity, and fibrin deposition in the ischemic penumbra provoked collateral failure and rapid expansion of the infarct core in mice with selective deficiency of S1P1 in ECs. This provides genetic evidence to support the critical and multifaceted neuroprotective roles of the endothelium in ischemic stroke and the importance of S1P signaling in coordinating these functions. Addressing mechanisms of S1P1 engagement, we uncover that the receptor is abluminally polarized and insensitive to circulating ligands in most ECs at the blood-neural barrier, imposing a need for cell-autonomous ligand provision for stroke protection and BBB penetration for supplemental therapeutic receptor engagement. This suggested optimal benefit of joint targeting of lymphocyte and, in particular, EC receptor pools with BBB-penetrating S1P1 agonists, a strategy that we demonstrate to limit cortical infarct spreading in mouse models of MCAO independent of reperfusion.
Our findings argue that endothelial S1P1 is dynamically engaged to limit infarct expansion, as neonatal deletion of S1P1 did not visibly alter vascular anatomy in the adult brain and as exacerbation of infarct size was also observed when S1P1 deficiency was induced in adulthood. Moreover, EC-selective deficiency in sphingosine kinases sensitized to MCAO to a similar degree as S1P1 deficiency even if it did not reproduce defects in retinal angiogenesis or in cerebrovascular homeostasis observed in S1pr1ECKO mice, and sensitization was overcome by compensating for the lack of endogenous ligand with a pharmacological S1P1 agonist.
S1P1 supports hallmark functions of the endothelium in (1) mediating smooth muscle relaxation, (2) maintaining vascular integrity, and (3) suppressing inflammation.7 We provide evidence that S1P1 exerts all these functions in the brain as follows:
1.
A role for EC S1P1 in control of cerebral blood flow in the naive brain was suggested by impaired acetylcholine- and hypercapnia-induced blood flow responses in S1pr1ECKO mice, impaired flow-mediated dilation in S1pr1ECKO cerebral arteries ex vivo, and the capacity of an S1P1 agonist to stimulate cerebral blood flow in wild-type but not in S1pr1ECKO mice. Impaired blood flow recovery and microvascular perfusion in S1pr1ECKO mice in the acute phase after MCAO argued that S1P1 also actively supports endothelial function during cerebral ischemia. This was substantiated by the impact of S1P1 deficiency on injury to the cortex, in which MCA branches are well connected to contiguous vascular territories, but not the striatum, in which they are not.28,49,50 S1P1 coordinates developmental angiogenesis by promoting perfusion of the nascent vasculature11 and supports flow-mediated dilation of mesenteric arteries.10 eNOS, which is important for the vasoactive functions of S1P1,10,11 also promotes the early establishment of collateral supply, thus counteracting infarct expansion in ischemic stroke.51 It is, therefore, likely that S1P1 acts at least in part, through eNOS. Blunted vasodilation in response to acetylcholine, hypercapnia, and flow, but not whisker stimulation, with EC S1P1 deficiency is intriguing and argues against a role for S1P1 in the mechanisms through which the endothelium contributes to retrograde propagation of vasoactive signals generated by neural activity. The dissociation between endothelium-dependent responses and functional hyperemia has also been observed in mice fed a high-salt diet, which display marked suppression of the eNOS-driven response to acetylcholine, but not of functional hyperemia.52,53 While the role of eNOS and other effectors downstream of S1P1 in this context remains to be specifically addressed, our results suggest that one important mechanism by which EC S1P1 limits infarct expansion is by facilitating retrograde perfusion of the affected MCA territory through collateral anastomoses with contiguous arterial branches.
2.
Further attesting to the importance of S1P1 in supporting the functions of the brain endothelium, naive S1pr1ECKO mice also displayed impaired BBB integrity. Although subtler in the naive brain than defects in lung vascular integrity observed in the same genetic model,9,39 dramatic and early onset edema after MCAO nevertheless argues for an important role for S1P1 in supporting BBB function during ischemia. Evans Blue/albumin leak more than doubled in S1pr1ECKO 24 hours after pMCAO, and magnetic resonance imaging revealed profound edema within hours after tMCAO. This suggests a possible role for S1P1 in regulating vesicular transport, as edema in ischemic stroke involves transcellular transport in the early phase and tight junction impairment in the late phase.40 Minimal impact of S1P1 deficiency on 4 kDa dextran leak in a model of septic encephalopathy in this study also argued against a critical role for S1P1 in maintaining tight junctions. This is consistent with a report showing that ApoM-S1P regulates vesicular transport in cerebral arterioles through S1P1 signaling.54 A second important mechanism by which EC S1P1 limits infarct expansion is, therefore, through the regulation of BBB integrity, suggesting that benefit afforded by S1P1 agonists on edema formation in ischemic and hemorrhagic stroke may involve direct actions on the BBB.6,21,24
3.
EC S1P1 deficiency was associated with increased ICAM-1 expression in the naive brain, suggestive of a role for S1P1 in suppressing endothelial activation and leukocyte adhesion. However, expression was not further increased in the context of ischemia, and we observed no significant difference in the early recruitment of neutrophils to the ischemic penumbra in S1pr1ECKO mice, nor in hemorrhagic transformation, which depends on leukocyte-mediated BBB destruction at later stages. Circulating markers of platelet activation and platelet recruitment to ischemic capillaries were also not substantially affected by EC S1P1 deficiency. However, the deposition of fibrin, which extended well beyond the boundary of no perfusion 3 hours after pMCAO, reached significantly further into distal MCA territories in S1pr1ECKO mice than in littermate controls. This suggests that microvascular coagulation contributes to rapid deterioration of collateral supply in S1pr1ECKO mice. Whether this reflects the loss of direct actions of S1P1 signaling on the anticoagulant or profibrinolytic status of the endothelium or is a consequence of reduced perfusion remains to be determined. Regardless, it highlights the critical dynamic role of the endothelium in maintaining microvascular patency.
Endothelial S1P1 signaling thus counteracts the expansion of the ischemic core by concerted actions on hallmark endothelial functions in the regulation of blood flow, BBB integrity, and microvascular patency.
Highly restricted S1P1 signaling in the naive cerebral cortex and the need for EC-autonomous S1P provision during cerebral ischemia both argue that S1P signaling is tightly controlled at the BBB. Characterization of S1P1 expression and signaling indicates that S1P1 may remain silent in most ECs after maturation of the blood-neural barrier due to S1P1 polarization away from circulating S1P in all but a subset of arteriolar ECs. As homeostatic functions of S1P1 in the naive brain did not all depend on EC S1P production, this subset of cells may sense circulating or other S1P sources. Although it is possible that the S1P1GS reporter mouse under-represents homeostatic S1P1 signaling, limited GFP accumulation in both ECs and perivascular cells suggests that brain ECs may export S1P only when stressed. In the resting state, most brain ECs express Sphk2, Spns2, and S1pr1, but not Sphk1.36 Induction of Sphk1 transcription as an acute response to ischemia observed in this study could thus represent a trigger for EC S1P1 activation in stroke and explain the limited effect of S1P1 deficiency observed immediately after MCA occlusion. High expression of lipid phosphate phosphatase-3 in pericytes, smooth muscle cells, and astrocytes of the neurovascular unit suggests that local S1P availability is also regulated by dephosphorylation.36,54 Limiting local S1P availability may serve both to maintain EC S1P1 responsiveness and to prevent S1P-mediated activation of astrocytes.55–57 Restricted EC S1P1 signaling also in peripheral organs suggests that polarization is not unique to the brain, although it has greater impact on the access of synthetic ligands at the BBB. Thus, homeostatic S1P1 signaling in the cerebral cortex appears to be maintained by a small subset of primarily arteriolar ECs that may have access to circulating ligand, while broader receptor engagement in ischemic stroke depends on the activation of EC-autonomous S1P production, possibly through the expression of Sphk1.
What are the implications of our study for therapeutic targeting of S1P1 in stroke? While modest protection observed in S1pr1HCKO mice in the tMCAO model supports the previously suggested benefit of targeting lymphocyte receptors,20 increased stroke severity and loss of efficacy of S1P1 agonists in S1pr1ECKO mice point to the endothelial receptor pool as their principal therapeutic target. This argues against the use of competitive antagonists or strong functional antagonists, which induce prolonged lymphopenia and may disable EC receptors. Most S1P1 agonists also induce lymphopenia, suggesting that these could provide benefit through both cellular targets. Our results argue that BBB penetration is needed to reach the EC receptor pool. FTY720, the S1P1 modulator employed in most experimental and clinical studies thus far, is nonspecific, did not efficiently penetrate the BBB in this study, desensitizes EC S1P1 at high doses, and induces long lasting lymphopenia.8,26,54 While FTY720 is probably mostly activating on EC S1P1 at therapeutic doses, our study argues that a more specific drug could be not only safer but also more efficacious. CYM-5442, an S1P1-selective agonist used in this study, is also desensitizing at high doses but rapidly distributes to the brain, triggers only transient immunosuppression, and is mostly washed out from plasma and brain 24 hours after administration.48 The suitability of other second-generation S1PR modulators needs to be defined. It is promising that recently Food and Drug Administration-approved Ozanimod and Siponimod both improve BBB function in models of intracerebral hemorrhage.21,24 New biased agonists have been developed that are minimally desensitizing on EC receptors and do not induce lymphopenia59,60; protection afforded with ApoM-Fc in a tMCAO model is consistent with our findings and suggest that targeting EC S1P1 alone may provide considerable benefit.59 The S1P1GS mouse used in this study and an analogous firefly split luciferase-based reporter provide valuable tools to assess BBB penetration of S1P1 agonists that elicit β-arrestin recruitment.38,61
In conclusion, this study supports a key role for S1P1 signaling in regulating endothelial function and vascular reactivity in the brain that expands with the engagement of EC-autonomous S1P provision to become a critical determinant of neuronal survival during cerebral ischemia. Although EC S1P1 signaling is partially sustained by cell-autonomous S1P provision, it can be boosted with BBB-penetrating pharmacological agonists. Joint targeting of lymphocyte20 and EC S1P1 with BBB-penetrating agonists is feasible and provides protection in transient and permanent ischemic stroke models in a therapeutically relevant time frame. Mechanistically, S1P1 targeting may promote regional blood flow, microvascular patency, and BBB integrity, independent of the prolonged immunosuppression induced by some S1P1 modulators.48 This is consistent with observed efficacy of FTY720 on downstream microvascular perfusion even in patients with failed recanalization to altapase22 and suggests that S1P1 agonists may preserve microvascular function and the recruitment of the collateral circulation even when recanalization is either not possible or not successful. This therapeutic strategy could, therefore, be envisioned in patients as soon as stroke is diagnosed, without waiting for the outcome of thrombolysis, which provides measurable benefit in <1/3 of patients treated.62 Sustained S1P1 expression during aging in mice (this study) and humans (Figure IX in the Data Supplement),63 and efficacy of S1P1 agonist on vascular parameters in murine models of diabetes and hypertension suggest that S1P1 remains a viable target in the typical stroke patient.10,64 This is underscored by efficacy of FTY720 in small-scale human trials for ischemic and hemorrhagic stroke, which also do not report an unexpected increase in bleeding, cardiac adverse events, or poststroke infections.22,23,65,66 Our findings suggest that this strategy may be refined with focus on minimally desensitizing BBB-penetrating S1P1 agonists. The critical protective roles for EC S1P1 and its mechanisms of engagement observed in this study may also be relevant to hemorrhagic stroke, vascular dementia, and ischemic disease of other organs.
Figure 3. Loss of endothelial cell (EC)-autonomous S1P1 (sphingosine 1-phosphate receptor-1) signaling does not impact cerebrovascular patterning. A, Isolectin B4 staining shows vascularization of the mouse retina at postnatal day (P)5 after neonatal Pdgfb iCreERT2-driven S1pr1 or Sphk1&2 deletion. Note delayed expansion of the vascular network (top, arrow) and abundant filopodia at the vascular front (bottom, arrowheads) of S1pr1ECKO but not Sphk1&2ECKO retinas. Scale bar: 1 mm (top), 50 μm (bottom) B, Quantification of outgrowth of the retinal vasculature at P5, normalized to weight of pups. C and D, Collateral connections between the middle cerebral artery (MCA) and branches of the posterior CA (PCA, white asterisk) and of the anterior CA (ACA, yellow asterisk) extending laterally from the midline between S1pr1ECKO and littermate controls. C, Quantification of ACA-MCA connections. D, Representative images. E, Vascular density in the cortex of S1pr1ECKO and littermate controls assessed by collagen IV (Coll IV) or Glut (Glucose transporter type)-1 staining. Representative images of Coll IV staining (left) and quantification of both (right), scale bar: 100 μm. Bar graphs show mean±SEM. Statistical significance assessed by 1-way ANOVA with Tukey multiple comparisons test (B) and unpaired t test (all other).
Figure 4. Endothelial S1P1 (sphingosine 1-phosphate receptor-1) sustains blood-brain barrier (BBB) function. A, Effect of Pdgfb-iCreERT2-mediated deletion of S1pr1 and Sphk1&2 on the accumulation of 4 kD TRITC-Dextran in the cerebral cortex of naive mice. B, Effect of Pdgfb-iCreERT2-mediated deletion of S1pr1 on the accumulation of 4 kD TRITC-Dextran in the cerebral cortex 8 h after challenge with 10 mg/kg lipopolysaccharide (LPS) intraperitoneal. C, Effect of Pdgfb-iCreERT2-mediated deletion (endothelial cell-selective knockout [ECKO]) of S1pr1 and Sphk1&2 as well as Mx1-Cre-mediated deletion (hematopoietic cell-selective knockout [HCKO]) of Sphk1&2 on the accumulation of Evans Blue/albumin in the cerebral cortex of naive mice. D, Effect of Pdgfb-iCreERT2-mediated deletion of S1pr1 on Evans Blue/albumin leak 24 h after permanent middle cerebral artery occlusion (pMCAO) in ipsilateral and contralateral hemispheres. Left, Representative brains. Right, Corrected absorbance of full hemisphere extracts. E, Full T2-weighted magnetic resonance imaging (MRI) 4 and 6 h after 90 min transient middle cerebral artery occlusion (tMCAO) in S1pr1ECKO and littermate controls. Left, Representative axial sections from level of the mid-olfactory bulb from the same animal at the 2 time points. Hatched line indicates midline, asterisk affected MCA territory, and arrow contralateral ventricle. Right, T2 lesion ratios calculated from MRI images based on axial plane images at the mid-olfactory bulb. Bar graphs show mean ± SEM. Statistical significance assessed by Mann-Whitney test (D) and unpaired t test (all other).
Figure 5. Endothelial S1P1 (sphingosine 1-phosphate receptor-1) regulates cerebral blood flow and supports tissue perfusion in the acute phase of stroke. A, Somatosensory cortex blood flow (CBF) assessed by laser Doppler flowmetry in mice equipped with a cranial window in response to whisker stimulation or superfusion of the endothelium-dependent vasodilator acetylcholine (ACh; 10 µmol/L) on exposed neocortex (left). Mean arterial blood pressures monitored in the femoral arteries during the CBF measurements (right). B, Mean blood flow velocities (mBFVs) measured by ultrasound in the basilar trunk of S1pr1ECKO mice and littermate controls before and 2–5 min after exposure to a gas mixture of 16% O2, 5% CO2, 79% N2 (normoxia-hypercapnia) and the relative change in velocities presented. Doppler image shows vessels analyzed in B and F. C, Flow-mediated dilation in posterior cerebral artery segments of S1pr1ECKO and littermate control mice assessed by arteriography. D, Blood pressures of nonsedated S1pr1ECKO and littermate control mice recorded for 72 h by telemetry. Average day and night systolic blood pressure (SBP) is shown; diastolic blood pressure and heart rates also did not differ between the groups. E, Basal perfusion of the cerebral cortex and hippocampus in S1pr1ECKO mice assessed by arterial spin labeling magnetic resonance imaging (MRI). F, mBFVs measured by ultrasound imaging in left and right intra cranial internal carotid artery (ICA) and basilar trunk (BT) under 0.5% isoflurane anesthesia before, 50 min and 2 h after electrocoagulation-induced permanent middle cerebral artery occlusion (pMCAO). Normalized values in ipsilateral ICA shown, absolute values for all arteries in Figure IIIC in the Data Supplement. G, mBFVs in the left ICA 2 h after left MCAO expressed as % of mean preocclusion velocities are plotted against infarct volumes in the same mice determined 24 h after occlusion. H, Blood flow in the leptomeningeal vasculature in the ipsilateral hemisphere was imaged by sidestream dark-field imaging through a cranial window 2–2.5 h after pMCAO. Left illustrates approximate positions of regions monitored at the MCA/anterior cerebral artery (ACA) border. Right shows results of automated analysis of microvascular perfusion in the MCA/ACA and MCA/posterior cerebral artery (PCA) border regions. Representative videos in Data Supplement. I, Infarct volumes 3 days after 35 min of transient middle cerebral artery occlusion (tMCAO) in S1pr1ECKO males and littermate controls. Graphs show mean±SEM. Statistical significance was assessed by repeated measures (C, D, and F) 2-way ANOVA with Bonferroni (C and F) or Sidak (D) multiple comparisons test, 1-way ANOVA (A CBF, E), linear regression analysis (G), Mann-Whitney test (H) or unpaired t test (all other). AzA indicates azygos artery.
Figure 6. Endothelial S1P1 (sphingosine 1-phosphate receptor-1) suppresses tissue perfusion and fibrin formation in middle cerebral artery (MCA) territories. A, ICAM (intercellular adhesion molecule)-1 protein in homogenates of cerebral cortex from naive S1pr1ECKO, Sphk1&2ECKO (Pdgfb-Cre) and Sphk1&2HCKO (Mx1-Cre) mice and littermate controls analyzed by Western blot and normalized to the vascular marker VE-Cadherin. B, ICAM-1 and MPO (myeloperoxidase) protein in homogenates of contra- and ipsilateral hemispheres 2.5 h after permanent MCA occlusion (pMCAO) from S1pr1ECKO mice and littermate controls. Top, Western Blot. Bottom, Quantification. C, Female S1pr1ECWT and S1pr1ECKO mice were analyzed 3 hours after pMCAO. Representative images of brain sections from S1pr1ECWT (left) and S1pr1ECKO (middle and right) mice. Dashed line indicates the perfusion border assessed by tomato lectin infusion 15 min before sacrifice (top). Fibrin(ogen) (red); endothelial cells (CD31, green), and platelets (CD41, blue/white). Square indicates area of higher magnification to the right. Note the extended area of poor perfusion superior to the infarct core and fibrin deposition both within and beyond the nonperfused regions. Scale bar: 500 µm for low magnification image and 100 µm for high magnification images. Lectin perfusion was assessed superior and inferior to the infarct core in an area of 600 µm×1200 µm (total), and fibrin(ogen) only superior to the infarct core in an area of 800 µm×1200 µm (total) or only within the distal part in an area of 800 µm×600 µm (distal). Bar graphs show mean±SEM. Statistical significance assessed by 1-way ANOVA with Holm-Sidak multiple comparisons test (B, left), Mann-Whitney (B, right) or unpaired t test (all other).
Figure 7. Receptor polarization restricts S1P1 (sphingosine 1-phosphate receptor-1) signaling and ligand access at the blood-neural barrier. A, S1P levels in plasma (left) and platelets (right) after permanent and transient filament-induced middle cerebral artery occlusion (MCAO) relative to sham. B–E, Assessment of S1P1 polarization in capillaries (B, C, and E) and artery (D) of the developing retina (B), adult retina (C and D) and adult cerebral cortex (E) of wild-type mice (B, C, D, E upper) and mice lacking S1pr1 in astrocytes (S1pr1ACK0; E middle) or in endothelial cells (ECs; S1pr1ECKO; E lower). Note that S1P1 expression (red) colocalizes with the luminal EC marker ICAM (intercellular adhesion molecule)-2 (green) in capillaries of the developing retina but not in the mature retina and brain. Luminal expression remains in some arterial ECs in the mature retina. The nonpolarized EC marker isolectin B4 (B and C) or Glut (Glucose transporter type)-1 (D and E) is shown in white, and the nuclear marker Hoechst in blue. Arrowheads indicate luminal and arrows abluminal side of the endothelium. Scale bars: 2 μm. Plots of fluorescence intensity are provided in Figure V in the Data Supplement. F, S1P1 signaling in the cerebral cortex studied in S1P1 GFP signaling reporter mice (S1P1GS) after systemic (0.6 mg/kg i.v.) or local (0.06 mg/kg intraparenchymal) administration of RP-001 or systemic FTY720 (2×5 mg/kg p.o.) or vehicle control by the same route. Note that signaling (GFP positive nuclei, green), usually restricted to ASMA positive arterioles (blue), is widespread in Erg-positive EC (red) after local administration of RP-001. GFP/Erg double-positive EC nuclei were counted in arterial (ASMA positive, lower) and other ECs (ASMA negative, upper) and expressed as a percentage of all ECs in the same category. Upper, Representative images; lower, image-based quantification (only statistical analysis for S1P1GS mice is shown and was done within and across treatment groups, respectively). Scale bar: 100 μm. GFP induction in other tissues in Figure VI in the Data Supplement, high-resolution image and quantification of non-EC GFP in Figure VII in the Data Supplement. G, Evolution of blood flow velocities in the cerebral cortex after injection of RP-001 i.v. (0.6 mg/kg; left) or into the cerebrospinal fluid (0.06 mg/kg; right) of S1pr1ECKO and littermate control mice. Error bars: SEM. H, S1P1 signaling in the cerebral cortex after systemic administration of CYM-5442 (10 mg/kg) or vehicle control. Left: Representative images, scale bar: 100 μm; right: Image-based quantification of GFP/Erg double-positive ECs as a fraction of total arterial and nonarterial ECs, respectively. Bar graphs show mean ± SEM. Statistical analyses by Mann-Whitney (F, local administration) or by 1- or 2-way ANOVA with Dunnett (A) or Tukey (all other) multiple comparisons test (all other).
Figure 8. A blood-brain barrier (BBB) penetrating S1P1 (sphingosine 1-phosphate receptor-1) agonist limits cortical infarct expansion in ischemic stroke. A, Effects of CYM-5442, RP-001, and FTY720 at indicated concentrations on lymphocyte (LY) counts 3 and 24 h after bolus administration. Values normalized to prebleed. Statistical significance assessed in comparison to vehicle control at indicated time points. B, Effect of CYM-5442 (3 mg/kg i.p. 0–6 h after occlusion) on infarct size 24 h after permanent middle cerebral artery occlusion (pMCAO) in wild-type males. C and D, Effect of CYM-5442 (3 mg/kg i.p. immediately after occlusion) on the impact of EC Sphk1&2 (C) and S1P1 (D) deficiency (Pdgfb-iCreERT2) on infarct size 24 h after pMCAO. E, Effect of CYM-5442 (3 mg/kg i.p. immediately before reperfusion) on infarct size 24 h after 60 min transient middle cerebral artery occlusion (tMCAO; left, representative infarcts; right, total and regional infarct size). F, Effect of RP-001 (0.6 mg/kg i.p. immediately after occlusion) on infarct size 24 h after pMCAO. G, Protective actions of CYM-5442 are accounted for mostly by engagement of endothelial S1P1, which promotes vasodilation and BBB integrity and limits fibrin deposition. By inducing transient lymphopenia through modulation of LY receptors, CYM-5442 may further reduce thromboinflammation, as has been previously described for FTY720. These distinct actions will act in concert to limit inflammation and edema and promote microvascular patency and perfusion of affected brain regions. Bar graphs show mean ± SEM. Statistical significance assessed by repeated measures 2-way ANOVA (A), Kruskall-Wallis test with Dunn multiple comparisons test (B), unpaired t test (C and F), or Mann-Whitney test (D and E). AC indicates astrocytes; Alb, albumin; PC, pericytes; PLT, platelet; and RBC, red blood cell.

Acknowledgments

We are grateful for input from all members of the Leducq SphingoNet network and for support from the technical and administrative platforms at the contributing institutions.

Novelty and Significance

VIEW EDITORIAL:New Vistas in Stroke Treatment

What Is Known?

•
Immune suppression and stimulation of vascular function have been investigated as adjunct therapies for the treatment of ischemic stroke.
•
S1P (Sphingosine 1-phosphate) signaling through S1P1 (S1P receptor-1) plays important roles in immune cell trafficking and vascular homeostasis.
•
S1P receptor modulators including fingolimod have shown promise for the treatment of ischemic and hemorrhagic stroke in both experimental models and small-scale clinical trials.
•
Mechanistically, these modulators have been considered to act principally by inhibiting S1P1-mediated sensing of circulating S1P, thus blocking lymphocyte trafficking and immunothrombosis.

What New Information Does This Article Contribute?

•
Disruption of homeostatic S1P1 signaling in the vascular endothelium, but not in lymphocytes, profoundly impacts outcome in mouse models of ischemic stroke.
•
Receptor polarization shields endothelial S1P1 from circulating endogenous and synthetic ligands and imposes a need for cell-autonomous S1P provision in the ischemic brain.
•
A blood-brain penetrating S1P1-selective agonist prevents infarct expansion in permanent and transient models of ischemic stroke when administered during a therapeutically relevant period of time after stroke onset.
Therapies for stroke are currently limited to mechanical and thrombolytic recanalization of the occluded artery. Immune suppression and stimulation of vascular function have both been explored as adjunct therapies to limit the expansion of the infarct core and reduce hemorrhagic transformation. S1P1 has emerged as a potential therapeutic target for both ischemic and hemorrhagic stroke, yet immunosuppression induced by S1P1 modulation could potentially increase the risk of poststroke infection. By revealing that the main protective action of S1P1 modulation depends on the endothelial receptor, we demonstrate that it is possible to obtain protection without the prolonged immunosuppression induced by some S1P1 modulators currently being explored for stroke therapy. Coupled with the need for blood-brain barrier penetration, this finding has implications as to the choice of S1P1 modulators for stroke therapy. Our study also highlights the potential of targeting endothelial function as a therapeutic approach for the treatment of ischemic stroke.

Footnote

Nonstandard Abbreviations and Acronyms

ACA
anterior cerebral artery
BBB
blood-brain barrier
EC
endothelial cell
eNOS
endothelial nitric oxide synthase
GPCR
G protein-coupled receptor
ICA
internal carotid artery
ICAM
intercellular adhesion molecule
MCA
middle cerebral artery
pMCAO
permanent middle cerebral artery occlusion
S1P
sphingosine 1-phosphate
S1P1
sphingosine 1-phosphate receptor-1
S1P1GS
S1P1 GFP signaling
tMCAO
transient middle cerebral artery occlusion
Spns2
spinster homolog 2

Supplemental Material

File (circres_circres-2020-316711_supp1.pdf)
File (circres_circres-2020-316711_supp4.pdf)
File (circres_circres-2020-316711_supp5.pdf)
File (circres_circres-2020-316711_supp6.mov)
File (circres_circres-2020-316711_supp7.mov)
File (circres_circres-2020-316711_supp8.mov)
File (nitzsche r3 supplement text 2020-12-15.pdf)
File (nitzsche r3 supplemental figures_201215_final_compressed_ec.pdf)

References

1.
Moskowitz MA, Lo EH, Iadecola C. The science of stroke: mechanisms in search of treatments. Neuron. 2010;67:181–198. doi: 10.1016/j.neuron.2010.07.002
Crossref
PubMed
Google Scholar
2.
Powers WJ, Rabinstein AA, Ackerson T, Adeoye OM, Bambakidis NC, Becker K, Biller J, Brown M, Demaerschalk BM, Hoh B, et al; American Heart Association Stroke Council. 2018 guidelines for the early management of patients with acute ischemic stroke: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2018;49:e46–e110. doi: 10.1161/STR.0000000000000158
Crossref
PubMed
Google Scholar
3.
Manning NW, Campbell BC, Oxley TJ, Chapot R. Acute ischemic stroke: time, penumbra, and reperfusion. Stroke. 2014;45:640–644. doi: 10.1161/STROKEAHA.113.003798
Crossref
PubMed
Google Scholar
4.
Bonnin P, Mazighi M, Charriaut-Marlangue C, Kubis N. Early collateral recruitment after stroke in infants and adults. Stroke. 2019;50:2604–2611. doi: 10.1161/STROKEAHA.119.025353
Crossref
PubMed
Google Scholar
5.
Shuaib A, Butcher K, Mohammad AA, Saqqur M, Liebeskind DS. Collateral blood vessels in acute ischaemic stroke: a potential therapeutic target. Lancet Neurol. 2011;10:909–921. doi: 10.1016/S1474-4422(11)70195-8
Crossref
PubMed
Google Scholar
6.
Dreikorn M, Milacic Z, Pavlovic V, Meuth SG, Kleinschnitz C, Kraft P. Immunotherapy of experimental and human stroke with agents approved for multiple sclerosis: a systematic review. Ther Adv Neurol Disord. 2018;11:1756286418770626. doi: 10.1177/1756286418770626
Crossref
Google Scholar
7.
Proia RL, Hla T. Emerging biology of sphingosine-1-phosphate: its role in pathogenesis and therapy. J Clin Invest. 2015;125:1379–1387. doi: 10.1172/JCI76369
Crossref
PubMed
Google Scholar
8.
Cyster JG, Schwab SR. Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs. Annu Rev Immunol. 2012;30:69–94. doi: 10.1146/annurev-immunol-020711-075011
Crossref
PubMed
Google Scholar
9.
Gazit SL, Mariko B, Thérond P, Decouture B, Xiong Y, Couty L, Bonnin P, Baudrie V, Le Gall SM, Dizier B, et al. Platelet and erythrocyte sources of S1P are redundant for vascular development and homeostasis, but both rendered essential after plasma S1P depletion in anaphylactic shock. Circ Res. 2016;119:e110–e126. doi: 10.1161/CIRCRESAHA.116.308929
Crossref
PubMed
Google Scholar
10.
Cantalupo A, Gargiulo A, Dautaj E, Liu C, Zhang Y, Hla T, Di Lorenzo A. S1PR1 (Sphingosine-1-Phosphate Receptor 1) signaling regulates blood flow and pressure. Hypertension. 2017;70:426–434. doi: 10.1161/HYPERTENSIONAHA.117.09088
Crossref
PubMed
Google Scholar
11.
Jung B, Obinata H, Galvani S, Mendelson K, Ding BS, Skoura A, Kinzel B, Brinkmann V, Rafii S, Evans T, et al. Flow-regulated endothelial S1P receptor-1 signaling sustains vascular development. Dev Cell. 2012;23:600–610. doi: 10.1016/j.devcel.2012.07.015
Crossref
PubMed
Google Scholar
12.
Galvani S, Sanson M, Blaho VA, Swendeman SL, Obinata H, Conger H, Dahlbäck B, Kono M, Proia RL, Smith JD, et al. HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation. Sci Signal. 2015;8:ra79. doi: 10.1126/scisignal.aaa2581
Crossref
PubMed
Google Scholar
13.
Pappu R, Schwab SR, Cornelissen I, Pereira JP, Regard JB, Xu Y, Camerer E, Zheng YW, Huang Y, Cyster JG, et al. Promotion of lymphocyte egress into blood and lymph by distinct sources of sphingosine-1-phosphate. Science. 2007;316:295–298. doi: 10.1126/science.1139221
Crossref
PubMed
Google Scholar
14.
Vu TM, Ishizu AN, Foo JC, Toh XR, Zhang F, Whee DM, Torta F, Cazenave-Gassiot A, Matsumura T, Kim S, et al. Mfsd2b is essential for the sphingosine-1-phosphate export in erythrocytes and platelets. Nature. 2017;550:524–528. doi: 10.1038/nature24053
Crossref
PubMed
Google Scholar
15.
Czech B, Pfeilschifter W, Mazaheri-Omrani N, Strobel MA, Kahles T, Neumann-Haefelin T, Rami A, Huwiler A, Pfeilschifter J. The immunomodulatory sphingosine 1-phosphate analog FTY720 reduces lesion size and improves neurological outcome in a mouse model of cerebral ischemia. Biochem Biophys Res Commun. 2009;389:251–256. doi: 10.1016/j.bbrc.2009.08.142
Crossref
PubMed
Google Scholar
16.
Wei Y, Yemisci M, Kim HH, Yung LM, Shin HK, Hwang SK, Guo S, Qin T, Alsharif N, Brinkmann V, et al. Fingolimod provides long-term protection in rodent models of cerebral ischemia. Ann Neurol. 2011;69:119–129. doi: 10.1002/ana.22186
Crossref
PubMed
Google Scholar
17.
Hasegawa Y, Suzuki H, Sozen T, Rolland W, Zhang JH. Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats. Stroke. 2010;41:368–374. doi: 10.1161/STROKEAHA.109.568899
Crossref
PubMed
Google Scholar
18.
Ichijo M, Ishibashi S, Li F, Yui D, Miki K, Mizusawa H, Yokota T. Sphingosine-1-phosphate receptor-1 selective agonist enhances collateral growth and protects against subsequent stroke. PLoS One. 2015;10:e0138029. doi: 10.1371/journal.pone.0138029
Crossref
PubMed
Google Scholar
19.
Brait VH, Tarrasón G, Gavaldà A, Godessart N, Planas AM. Selective sphingosine 1-phosphate receptor 1 agonist is protective against ischemia/reperfusion in mice. Stroke. 2016;47:3053–3056. doi: 10.1161/STROKEAHA.116.015371
Crossref
PubMed
Google Scholar
20.
Kraft P, Göb E, Schuhmann MK, Göbel K, Deppermann C, Thielmann I, Herrmann AM, Lorenz K, Brede M, Stoll G, et al. FTY720 ameliorates acute ischemic stroke in mice by reducing thrombo-inflammation but not by direct neuroprotection. Stroke. 2013;44:3202–3210. doi: 10.1161/STROKEAHA.113.002880
Crossref
PubMed
Google Scholar
21.
Sun N, Shen Y, Han W, Shi K, Wood K, Fu Y, Hao J, Liu Q, Sheth KN, Huang D, et al. Selective sphingosine-1-phosphate receptor 1 modulation attenuates experimental intracerebral hemorrhage. Stroke. 2016;47:1899–1906. doi: 10.1161/STROKEAHA.115.012236
Crossref
PubMed
Google Scholar
22.
Tian DC, Shi K, Zhu Z, Yao J, Yang X, Su L, Zhang S, Zhang M, Gonzales RJ, Liu Q, et al. Fingolimod enhances the efficacy of delayed alteplase administration in acute ischemic stroke by promoting anterograde reperfusion and retrograde collateral flow. Ann Neurol. 2018;84:717–728. doi: 10.1002/ana.25352
Crossref
PubMed
Google Scholar
23.
Zhu Z, Fu Y, Tian D, Sun N, Han W, Chang G, Dong Y, Xu X, Liu Q, Huang D, et al. Combination of the immune modulator fingolimod with alteplase in acute ischemic stroke: a pilot trial. Circulation. 2015;132:1104–1112. doi: 10.1161/CIRCULATIONAHA.115.016371
Crossref
PubMed
Google Scholar
24.
Bobinger T, Manaenko A, Burkardt P, Beuscher V, Sprügel MI, Roeder SS, Bäuerle T, Seyler L, Nagel AM, Linker RA, et al. Siponimod (BAF-312) attenuates perihemorrhagic edema and improves survival in experimental intracerebral hemorrhage. Stroke. 2019;50:3246–3254.
Crossref
PubMed
Google Scholar
25.
Niazi H, Zoghdani N, Couty L, Leuci A, Nitzsche A, Allende ML, Mariko B, Ishaq R, Aslan Y, Becker PH, et al. Murine platelet production is suppressed by S1P release in the hematopoietic niche, not facilitated by blood S1P sensing. Blood Adv. 2019;3:1702–1713. doi: 10.1182/bloodadvances.2019031948
Crossref
PubMed
Google Scholar
26.
Yanagida K, Liu CH, Faraco G, Galvani S, Smith HK, Burg N, Anrather J, Sanchez T, Iadecola C, Hla T. Size-selective opening of the blood-brain barrier by targeting endothelial sphingosine 1-phosphate receptor 1. Proc Natl Acad Sci USA. 2017;114:4531–4536. doi: 10.1073/pnas.1618659114
Crossref
PubMed
Google Scholar
27.
Poittevin M, Deroide N, Azibani F, Delcayre C, Giannesini C, Levy BI, Pocard M, Kubis N. Glatiramer acetate administration does not reduce damage after cerebral ischemia in mice. J Neuroimmunol. 2013;254:55–62. doi: 10.1016/j.jneuroim.2012.09.009
Crossref
PubMed
Google Scholar
28.
Prinz, V, Endres, M. Modeling focal cerebral ischemia in rodents: Introduction and overview. Dirnagl, U., ed. In: Rodent models of stroke. Humana Press: Springer Protocols; 2016:19–30.
Google Scholar
29.
Claxton S, Kostourou V, Jadeja S, Chambon P, Hodivala-Dilke K, Fruttiger M. Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis. 2008;46:74–80. doi: 10.1002/dvg.20367
Crossref
PubMed
Google Scholar
30.
Zhang Y, Jin Y, Behr MJ, Feustel PJ, Morrison JP, Kimelberg HK. Behavioral and histological neuroprotection by tamoxifen after reversible focal cerebral ischemia. Exp Neurol. 2005;196:41–46. doi: 10.1016/j.expneurol.2005.07.002
Crossref
PubMed
Google Scholar
31.
Wakade C, Khan MM, De Sevilla LM, Zhang QG, Mahesh VB, Brann DW. Tamoxifen neuroprotection in cerebral ischemia involves attenuation of kinase activation and superoxide production and potentiation of mitochondrial superoxide dismutase. Endocrinology. 2008;149:367–379. doi: 10.1210/en.2007-0899
Crossref
PubMed
Google Scholar
32.
Camerer E, Regard JB, Cornelissen I, Srinivasan Y, Duong DN, Palmer D, Pham TH, Wong JS, Pappu R, Coughlin SR. Sphingosine-1-phosphate in the plasma compartment regulates basal and inflammation-induced vascular leak in mice. J Clin Invest. 2009;119:1871–1879. doi: 10.1172/jci38575
Crossref
PubMed
Google Scholar
33.
Kim GS, Yang L, Zhang G, Zhao H, Selim M, McCullough LD, Kluk MJ, Sanchez T. Critical role of sphingosine-1-phosphate receptor-2 in the disruption of cerebrovascular integrity in experimental stroke. Nat Commun. 2015;6:7893. doi: 10.1038/ncomms8893
Crossref
PubMed
Google Scholar
34.
Kühn R, Schwenk F, Aguet M, Rajewsky K. Inducible gene targeting in mice. Science. 1995;269:1427–1429. doi: 10.1126/science.7660125
Crossref
PubMed
Google Scholar
35.
Frej C, Andersson A, Larsson B, Guo LJ, Norström E, Happonen KE, Dahlbäck B. Quantification of sphingosine 1-phosphate by validated LC-MS/MS method revealing strong correlation with apolipoprotein M in plasma but not in serum due to platelet activation during blood coagulation. Anal Bioanal Chem. 2015;407:8533–8542. doi: 10.1007/s00216-015-9008-4
Crossref
PubMed
Google Scholar
36.
Vanlandewijck M, He L, Mäe MA, Andrae J, Ando K, Del Gaudio F, Nahar K, Lebouvier T, Laviña B, Gouveia L, et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature. 2018;554:475–480. doi: 10.1038/nature25739
Crossref
PubMed
Google Scholar
37.
Ximerakis M, Lipnick SL, Innes BT, Simmons SK, Adiconis X, Dionne D, Mayweather BA, Nguyen L, Niziolek Z, Ozek C, et al. Single-cell transcriptomic profiling of the aging mouse brain. Nat Neurosci. 2019;22:1696–1708. doi: 10.1038/s41593-019-0491-3
Crossref
PubMed
Google Scholar
38.
Kono M, Tucker AE, Tran J, Bergner JB, Turner EM, Proia RL. Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo. J Clin Invest. 2014;124:2076–2086. doi: 10.1172/JCI71194
Crossref
PubMed
Google Scholar
39.
Blaho VA, Galvani S, Engelbrecht E, Liu C, Swendeman SL, Kono M, Proia RL, Steinman L, Han MH, Hla T. HDL-bound sphingosine-1-phosphate restrains lymphopoiesis and neuroinflammation. Nature. 2015;523:342–346. doi: 10.1038/nature14462
Crossref
PubMed
Google Scholar
40.
Knowland D, Arac A, Sekiguchi KJ, Hsu M, Lutz SE, Perrino J, Steinberg GK, Barres BA, Nimmerjahn A, Agalliu D. Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke. Neuron. 2014;82:603–617. doi: 10.1016/j.neuron.2014.03.003
Crossref
PubMed
Google Scholar
41.
Jackman KA, Zhou P, Faraco G, Peixoto PM, Coleman C, Voss HU, Pickel V, Manfredi G, Iadecola C. Dichotomous effects of chronic intermittent hypoxia on focal cerebral ischemic injury. Stroke. 2014;45:1460–1467. doi: 10.1161/STROKEAHA.114.004816
Crossref
PubMed
Google Scholar
42.
Desilles JP, Syvannarath V, Di Meglio L, Ducroux C, Boisseau W, Louedec L, Jandrot-Perrus M, Michel JB, Mazighi M, Ho-Tin-Noe B. Downstream microvascular thrombosis in cortical venules is an early response to proximal cerebral arterial occlusion. J Am Heart Assoc. 2018;7:e007804.
Crossref
PubMed
Google Scholar
43.
von Brühl ML, Stark K, Steinhart A, Chandraratne S, Konrad I, Lorenz M, Khandoga A, Tirniceriu A, Coletti R, Köllnberger M, et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med. 2012;209:819–835. doi: 10.1084/jem.20112322
Crossref
PubMed
Google Scholar
44.
Knapp M, Zendzian-Piotrowska M, Błachnio-Zabielska A, Zabielski P, Kurek K, Górski J. Myocardial infarction differentially alters sphingolipid levels in plasma, erythrocytes and platelets of the rat. Basic Res Cardiol. 2012;107:294. doi: 10.1007/s00395-012-0294-0
Crossref
PubMed
Google Scholar
45.
Cahalan SM, Gonzalez-Cabrera PJ, Sarkisyan G, Nguyen N, Schaeffer MT, Huang L, Yeager A, Clemons B, Scott F, Rosen H. Actions of a picomolar short-acting S1P1 agonist in S1P1-eGFP knock-in mice. Nat Chem Biol. 2011;7:254–256. doi: 10.1038/nchembio.547
Crossref
PubMed
Google Scholar
46.
Rangroo Thrane V, Thrane AS, Plog BA, Thiyagarajan M, Iliff JJ, Deane R, Nagelhus EA, Nedergaard M. Paravascular microcirculation facilitates rapid lipid transport and astrocyte signaling in the brain. Sci Rep. 2013;3:2582. doi: 10.1038/srep02582
Crossref
PubMed
Google Scholar
47.
Foster CA, Howard LM, Schweitzer A, Persohn E, Hiestand PC, Balatoni B, Reuschel R, Beerli C, Schwartz M, Billich A. Brain penetration of the oral immunomodulatory drug FTY720 and its phosphorylation in the central nervous system during experimental autoimmune encephalomyelitis: consequences for mode of action in multiple sclerosis. J Pharmacol Exp Ther. 2007;323:469–475. doi: 10.1124/jpet.107.127183
Crossref
PubMed
Google Scholar
48.
Gonzalez-Cabrera PJ, Cahalan SM, Nguyen N, Sarkisyan G, Leaf NB, Cameron MD, Kago T, Rosen H. S1P(1) receptor modulation with cyclical recovery from lymphopenia ameliorates mouse model of multiple sclerosis. Mol Pharmacol. 2012;81:166–174. doi: 10.1124/mol.111.076109
Crossref
PubMed
Google Scholar
49.
Zhang H, Prabhakar P, Sealock R, Faber JE. Wide genetic variation in the native pial collateral circulation is a major determinant of variation in severity of stroke. J Cereb Blood Flow Metab. 2010;30:923–934. doi: 10.1038/jcbfm.2010.10
Crossref
PubMed
Google Scholar
50.
Seyman E, Shaim H, Shenhar-Tsarfaty S, Jonash-Kimchi T, Bornstein NM, Hallevi H. The collateral circulation determines cortical infarct volume in anterior circulation ischemic stroke. BMC Neurol. 2016;16:206. doi: 10.1186/s12883-016-0722-0
Crossref
PubMed
Google Scholar
51.
Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, Moskowitz MA. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. J Cereb Blood Flow Metab. 1996;16:981–987. doi: 10.1097/00004647-199609000-00023
Crossref
PubMed
Google Scholar
52.
Faraco G, Brea D, Garcia-Bonilla L, Wang G, Racchumi G, Chang H, Buendia I, Santisteban MM, Segarra SG, Koizumi K, et al. Dietary salt promotes neurovascular and cognitive dysfunction through a gut-initiated TH17 response. Nat Neurosci. 2018;21:240–249. doi: 10.1038/s41593-017-0059-z
Crossref
PubMed
Google Scholar
53.
Faraco G, Hochrainer K, Segarra SG, Schaeffer S, Santisteban MM, Menon A, Jiang H, Holtzman DM, Anrather J, Iadecola C. Dietary salt promotes cognitive impairment through tau phosphorylation. Nature. 2019;574:686–690. doi: 10.1038/s41586-019-1688-z
Crossref
PubMed
Google Scholar
54.
Mathiesen Janiurek M, Soylu-Kucharz R, Christoffersen C, Kucharz K, Lauritzen M. Apolipoprotein M-bound sphingosine-1-phosphate regulates blood-brain barrier paracellular permeability and transcytosis. Elife. 2019;8:e49405.
Crossref
PubMed
Google Scholar
55.
Gómez-López S, Martínez-Silva AV, Montiel T, Osorio-Gómez D, Bermúdez-Rattoni F, Massieu L, Escalante-Alcalde D. Neural ablation of the PARK10 candidate Plpp3 leads to dopaminergic transmission deficits without neurodegeneration. Sci Rep. 2016;6:24028. doi: 10.1038/srep24028
Crossref
PubMed
Google Scholar
56.
Choi JW, Gardell SE, Herr DR, Rivera R, Lee CW, Noguchi K, Teo ST, Yung YC, Lu M, Kennedy G, et al. FTY720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation. Proc Natl Acad Sci USA. 2011;108:751–756. doi: 10.1073/pnas.1014154108
Crossref
PubMed
Google Scholar
57.
Gril B, Paranjape AN, Woditschka S, Hua E, Dolan EL, Hanson J, Wu X, Kloc W, Izycka-Swieszewska E, Duchnowska R, et al. Reactive astrocytic S1P3 signaling modulates the blood-tumor barrier in brain metastases. Nat Commun. 2018;9:2705. doi: 10.1038/s41467-018-05030-w
Crossref
PubMed
Google Scholar
58.
Oo ML, Chang SH, Thangada S, Wu MT, Rezaul K, Blaho V, Hwang SI, Han DK, Hla T. Engagement of S1P1-degradative mechanisms leads to vascular leak in mice. J Clin Invest. 2011;121:2290–2300. doi: 10.1172/JCI45403
Crossref
PubMed
Google Scholar
59.
Swendeman SL, Xiong Y, Cantalupo A, Yuan H, Burg N, Hisano Y, Cartier A, Liu CH, Engelbrecht E, Blaho V, et al. An engineered S1P chaperone attenuates hypertension and ischemic injury. Sci Signal. 2017;10:eaal2722.
Crossref
PubMed
Google Scholar
60.
Poirier B, Briand V, Kadereit D, Schafer M, Wohlfart P, Philippo MC, Caillaud D, Gouraud L, Grailhe P, Bidouard JP, et al. A G protein-biased S1P1 agonist, SAR247799, protects endothelial cells without affecting lymphocyte numbers. Sci Signal. 2020;13:eaax8050.
Crossref
PubMed
Google Scholar
61.
Kono M, Conlon EG, Lux SY, Yanagida K, Hla T, Proia RL. Bioluminescence imaging of G protein-coupled receptor activation in living mice. Nat Commun. 2017;8:1163. doi: 10.1038/s41467-017-01340-7
Crossref
PubMed
Google Scholar
62.
Saver JL, Gornbein J, Grotta J, Liebeskind D, Lutsep H, Schwamm L, Scott P, Starkman S. Number needed to treat to benefit and to harm for intravenous tissue plasminogen activator therapy in the 3- to 4.5-hour window: joint outcome table analysis of the ECASS 3 trial. Stroke. 2009;40:2433–2437. doi: 10.1161/STROKEAHA.108.543561
Crossref
PubMed
Google Scholar
63.
Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson Å, Kampf C, Sjöstedt E, Asplund A, et al. Proteomics. Tissue-based map of the human proteome. Science. 2015;347:1260419. doi: 10.1126/science.1260419
Crossref
PubMed
Google Scholar
64.
Whetzel AM, Bolick DT, Srinivasan S, Macdonald TL, Morris MA, Ley K, Hedrick CC. Sphingosine-1 phosphate prevents monocyte/endothelial interactions in type 1 diabetic NOD mice through activation of the S1P1 receptor. Circ Res. 2006;99:731–739. doi: 10.1161/01.RES.0000244088.33375.52
Crossref
PubMed
Google Scholar
65.
Fu Y, Zhang N, Ren L, Yan Y, Sun N, Li YJ, Han W, Xue R, Liu Q, Hao J, et al. Impact of an immune modulator fingolimod on acute ischemic stroke. Proc Natl Acad Sci U S A. 2014;111:18315–18320.
Crossref
PubMed
Google Scholar
66.
Li YJ, Shi SX, Liu Q, Shi FD, Gonzales RJ. Targeted role for sphingosine-1-phosphate receptor 1 in cerebrovascular integrity and inflammation during acute ischemic stroke. Neurosci Lett. 2020;735:135160.
Crossref
PubMed
Google Scholar
67.
Pitulescu ME, Adams RH. Eph/ephrin molecules–a hub for signaling and endocytosis. Genes Dev. 2010;24:2480–2492. doi: 10.1101/gad.1973910
Crossref
PubMed
Google Scholar
68.
Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45:593–605. doi: 10.1002/dvg.20335
Crossref
PubMed
Google Scholar
69.
Bonnin P, Leger PL, Deroide N, Fau S, Baud O, Pocard M, Charriaut-Marlangue C, Renolleau S. Impact of intracranial blood-flow redistribution on stroke size during ischemia-reperfusion in 7-day-old rats. J Neurosci Methods. 2011;198:103–109. doi: 10.1016/j.jneumeth.2011.02.030
Crossref
PubMed
Google Scholar
70.
Poittevin M, Bonnin P, Pimpie C, Rivière L, Sebrié C, Dohan A, Pocard M, Charriaut-Marlangue C, Kubis N. Diabetic microangiopathy: impact of impaired cerebral vasoreactivity and delayed angiogenesis after permanent middle cerebral artery occlusion on stroke damage and cerebral repair in mice. Diabetes. 2015;64:999–1010. doi: 10.2337/db14-0759
Crossref
PubMed
Google Scholar
71.
Faraco G, Moraga A, Moore J, Anrather J, Pickel VM, Iadecola C. Circulating endothelin-1 alters critical mechanisms regulating cerebral microcirculation. Hypertension. 2013;62:759–766. doi: 10.1161/HYPERTENSIONAHA.113.01761
Crossref
PubMed
Google Scholar
72.
Garcia-Bonilla L, Racchumi G, Murphy M, Anrather J, Iadecola C. Endothelial CD36 contributes to postischemic brain injury by promoting neutrophil activation via CSF3. J Neurosci. 2015;35:14783–14793. doi: 10.1523/JNEUROSCI.2980-15.2015
Crossref
PubMed
Google Scholar
73.
Jackman K, Kunz A, Iadecola C. Modeling focal cerebral ischemia in vivo. Methods Mol Biol. 2011;793:195–209. doi: 10.1007/978-1-61779-328-8_13
Crossref
PubMed
Google Scholar
74.
Leger PL, Bonnin P, Moretti R, Tanaka S, Duranteau J, Renolleau S, Baud O, Charriaut-Marlangue C. Early recruitment of cerebral microcirculation by neuronal nitric oxide synthase inhibition in a Juvenile Ischemic Rat Model. Cerebrovasc Dis. 2016;41:40–49. doi: 10.1159/000441663
Crossref
PubMed
Google Scholar
75.
Henrion D, Dechaux E, Dowell FJ, Maclour J, Samuel JL, Lévy BI, Michel JB. Alteration of flow-induced dilatation in mesenteric resistance arteries of L-NAME treated rats and its partial association with induction of cyclo-oxygenase-2. Br J Pharmacol. 1997;121:83–90. doi: 10.1038/sj.bjp.0701109
Crossref
PubMed
Google Scholar
76.
Franco CA, Jones ML, Bernabeu MO, Geudens I, Mathivet T, Rosa A, Lopes FM, Lima AP, Ragab A, Collins RT, et al. Dynamic endothelial cell rearrangements drive developmental vessel regression. PLoS Biol. 2015;13:e1002125. doi: 10.1371/journal.pbio.1002125
Crossref
PubMed
Google Scholar
77.
Lamprecht MR, Sabatini DM, Carpenter AE. CellProfiler: free, versatile software for automated biological image analysis. Biotechniques. 2007;42:71–75. doi: 10.2144/000112257
Crossref
PubMed
Google Scholar
78.
Lee YK, Uchida H, Smith H, Ito A, Sanchez T. The isolation and molecular characterization of cerebral microvessels. Nat Protoc. 2019;14:3059–3081. doi: 10.1038/s41596-019-0212-0
Crossref
PubMed
Google Scholar
79.
Ruck T, Bittner S, Epping L, Herrmann AM, Meuth SG. Isolation of primary murine brain microvascular endothelial cells. J Vis Exp. 2014;93:e52204.
PubMed
Google Scholar
80.
Regard JB, Sato IT, Coughlin SR. Anatomical profiling of G protein-coupled receptor expression. Cell. 2008;135:561–571. doi: 10.1016/j.cell.2008.08.040
Crossref
PubMed
Google Scholar
81.
Assmann JC, Muller K, Wenzel J, Walther T, Brands J, Thornton P, Allan SM, Schwaninger M. Isolation and cultivation of primary brain endothelial cells from adult mice. Bio Protoc. 2017;7:e2294.
Crossref
PubMed
Google Scholar
82.
He L, Vanlandewijck M, Mäe MA, Andrae J, Ando K, Del Gaudio F, Nahar K, Lebouvier T, Laviña B, Gouveia L, et al. Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types. Sci Data. 2018;5:180160. doi: 10.1038/sdata.2018.160
Crossref
PubMed
Google Scholar

eLetters(0)

eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.

Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.

Sign In to Submit a Response to This Article

Information & Authors

Information

Published In

Go to Circulation Research
Go to Circulation Research
Circulation Research
Volume 128 • Number 3 • 5 February 2021
Pages: 363 - 382
PubMed: 33301355

Copyright

© 2020 American Heart Association, Inc.

Versions

You are viewing the most recent version of this article.

History

Received: 24 January 2020
Revision received: 12 November 2020
Accepted: 1 December 2020
Published online: 2 December 2020
Published in print: 5 February 2021

Permissions

Request permissions for this article.

Keywords

  1. blood-brain barrier
  2. collateral circulation
  3. endothelium
  4. fingolimod hydrochloride
  5. neuroprotective agents
  6. stroke

Subjects

  1. Cerebrovascular Disease/Stroke
  2. Hemodynamics
  3. Ischemic Stroke
  4. Pharmacology
  5. Vascular Biology

Authors

Affiliations

Anja Nitzsche* https://orcid.org/0000-0003-0567-6790
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Marine Poittevin*
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
Institut des Vaisseaux et du Sang, Hôpital Lariboisière, France (M.P., P.-L.L.).
View all articles by this author
Ammar Benarab*
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Philippe Bonnin* https://orcid.org/0000-0003-3184-3740
Université de Paris, INSERM U965 and Physiologie Clinique - Explorations-Fonctionnelles, AP-HP, Hôpital Lariboisière, France (P.B., N.K.).
View all articles by this author
Giuseppe Faraco https://orcid.org/0000-0001-8675-4580
Feil Family Brain and Mind Research Institute (G.F., L.G.-B., C.I.), Weill Cornell Medical College, Cornell University, New York.
View all articles by this author
Hiroki Uchida
Center for Vascular Biology (H.U., T.S.), Weill Cornell Medical College, Cornell University, New York.
View all articles by this author
Julie Favre
MITOVASC Institute, CARFI Facility, CNRS UMR 6015, INSERM U1083, Angers University, France (J.F., M.C.L.G., D.H.).
View all articles by this author
Lidia Garcia-Bonilla
Feil Family Brain and Mind Research Institute (G.F., L.G.-B., C.I.), Weill Cornell Medical College, Cornell University, New York.
View all articles by this author
Manuela C.L. Garcia
MITOVASC Institute, CARFI Facility, CNRS UMR 6015, INSERM U1083, Angers University, France (J.F., M.C.L.G., D.H.).
View all articles by this author
Pierre-Louis Léger
Institut des Vaisseaux et du Sang, Hôpital Lariboisière, France (M.P., P.-L.L.).
INSERM U1141, Hôpital Robert Debré (P.-L.L., C.C.-M.).
View all articles by this author
Patrice Thérond
Assistance Publique-Hôpitaux de Paris (AP-HP), Service de Biochimie, Hôpital de Bicêtre, Le Kremlin Bicêtre, France (P.T.).
Université Paris-Sud, France (P.T.).
UFR de Pharmacie, EA 4529, Châtenay-Malabry, France (P.T.).
View all articles by this author
Thomas Mathivet https://orcid.org/0000-0001-7761-1684
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Gwennhael Autret https://orcid.org/0000-0001-7335-350X
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Véronique Baudrie
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Ludovic Couty
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Mari Kono https://orcid.org/0000-0003-2447-4350
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Institutes of Health, Bethesda, MD, USA (M.K., R.L.P.).
View all articles by this author
Hira Niazi https://orcid.org/0000-0003-4737-2613
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Pierre-Louis Tharaux https://orcid.org/0000-0002-6062-5905
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Jerold Chun
Neuroscience Drug Discovery, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA (J.C.).
View all articles by this author
Susan R. Schwab
Skirball Institute of Biomolecular Medicine, New York University School of Medicine, NY (S.R.S.).
View all articles by this author
Anne Eichmann
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Bertrand Tavitian https://orcid.org/0000-0002-5349-8194
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author
Richard L. Proia
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Institutes of Health, Bethesda, MD, USA (M.K., R.L.P.).
View all articles by this author
Christiane Charriaut-Marlangue https://orcid.org/0000-0002-9632-5453
INSERM U1141, Hôpital Robert Debré (P.-L.L., C.C.-M.).
View all articles by this author
Teresa Sanchez https://orcid.org/0000-0002-2711-7423
Center for Vascular Biology (H.U., T.S.), Weill Cornell Medical College, Cornell University, New York.
View all articles by this author
Nathalie Kubis
Université de Paris, INSERM U965 and Physiologie Clinique - Explorations-Fonctionnelles, AP-HP, Hôpital Lariboisière, France (P.B., N.K.).
Université de Paris, INSERM U1148, Hôpital Bichat, France (N.K.).
View all articles by this author
Daniel Henrion https://orcid.org/0000-0003-1094-0285
MITOVASC Institute, CARFI Facility, CNRS UMR 6015, INSERM U1083, Angers University, France (J.F., M.C.L.G., D.H.).
View all articles by this author
Costantino Iadecola https://orcid.org/0000-0001-9797-073X
Feil Family Brain and Mind Research Institute (G.F., L.G.-B., C.I.), Weill Cornell Medical College, Cornell University, New York.
View all articles by this author
Timothy Hla https://orcid.org/0000-0001-8355-4065
Vascular Biology Program, Boston Children’s Hospital, MA (T.H.).
View all articles by this author
Eric Camerer https://orcid.org/0000-0002-6271-7125 [email protected]
Université de Paris, Paris Cardiovascular Research Centre, INSERM U970, France (A.N., M.P., A.B., T.M., G.A., V.B., L.C., A.C., H.N., P.-L.T., A.E., B.T., E.C.).
View all articles by this author

Notes

*
A.N., M.P., A.B., and P.B. contributed equally to this article.
The Data Supplement is available with this article at Supplemental Material.
For Sources of Funding and Disclosures, see page 380.
Correspondence to: Eric Camerer, Université de Paris, PARCC, INSERM U970, 56 Rue Leblanc, F-75015 Paris, France. Email [email protected]

Disclosures

Disclosures None.

Sources of Funding

This work was supported by Fondation Leducq (SphingoNet; E. Camerer, T. Sanchez, R.L. Proia, C. Iadecola, and T. Hla), Fondation pour la Recherche Medicale (DCP20171138945; E. Camerer, A. Eichmann, and B. Tavitian), the French National Research Agency (ANR-19-CE14-0028; E. Camerer, D. Henrion, and P. Bonnin), Fondation de France (E. Camerer), Marie Curie Actions (PRESTIGE-2016-3-0011; A. Nitzsche), Lefoulon Delalande (A. Chevallier and A. Nitzsche), the Intramural Research Program of the National Institutes of Health, the National Institute of Diabetes and Digestive and Kidney Diseases (R.L. Proia), National Institutes of Health, National Institute of Neurological Disorders and Stroke (NS114561; T. Sanchez) and Fondation Grace de Monaco (P.-L. Léger and C. Charriaut-Marlangue).

Metrics & Citations

Metrics

Citations

Download Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Select your manager software from the list below and click Download.

    • Yilun Ma,
    • Taiwei Dong,
    • Fei Luan,
    • Juanjuan Yang,
    • Feng Miao,
    • Peifeng Wei,
    Interaction of major facilitator superfamily domain containing 2A with the blood–brain barrier, Neural Regeneration Research, 20, 8, (2133-2152), (2024).https://doi.org/10.4103/NRR.NRR-D-24-00191
    Crossref
    • Jian Duan,
    • Qijie Zhao,
    • Zeyuan He,
    • Shuang Tang,
    • Jia Duan,
    • Wenli Xing,
    Current understanding of macrophages in intracranial aneurysm: relevant etiological manifestations, signaling modulation and therapeutic strategies, Frontiers in Immunology, 14, (2024).https://doi.org/10.3389/fimmu.2023.1320098
    Crossref
    • Junjie Zhang,
    • Qingyu Ge,
    • Tianpeng Du,
    • Yuhao Kuang,
    • Zongyao Fan,
    • Xinyi Jia,
    • Wenjin Gu,
    • Zhengsen Chen,
    • Zhongqing Wei,
    • Baixin Shen,
    SPHK1/S1PR1/PPAR-α axis restores TJs between uroepithelium providing new ideas for IC/BPS treatment, Life Science Alliance, 8, 2, (e202402957), (2024).https://doi.org/10.26508/lsa.202402957
    Crossref
    • 保云 金,
    Construction of Plasma Sphingosine 1-Phosphate Expression Level and Risk Nomogram in Patients with Ischemic Stroke, Advances in Clinical Medicine, 14, 05, (901-910), (2024).https://doi.org/10.12677/acm.2024.1451505
    Crossref
    • Zhonghao Li,
    • Wei Wu,
    • Qiushi Li,
    • Xin Heng,
    • Wei Zhang,
    • Yinghong Zhu,
    • Lin Chen,
    • Ziqi Chen,
    • Mengcheng Shen,
    • Ning Ma,
    • Qingzhong Xiao,
    • Yi Yan,
    BCL6B-dependent suppression of ETV2 hampers endothelial cell differentiation, Stem Cell Research & Therapy, 15, 1, (2024).https://doi.org/10.1186/s13287-024-03832-y
    Crossref
    • Nina Hagemann,
    • Yachao Qi,
    • Ayan Mohamud Yusuf,
    • AnRan Li,
    • Xiaoni Zhang,
    • Philippa Spangenberg,
    • Anthony Squire,
    • Thorsten R Doeppner,
    • Fengyan Jin,
    • Shuo Zhao,
    • Jianxu Chen,
    • Axel Mosig,
    • Matthias Gunzer,
    • Dirk M Hermann,
    Arterial specification precedes microvascular restitution in the peri-infarct cortex that is driven by small microvessels, Journal of Cerebral Blood Flow & Metabolism, (2024).https://doi.org/10.1177/0271678X241270407
    Crossref
    • Nina Hagemann,
    • Yachao Qi,
    • Ayan Mohamud Yusuf,
    • AnRan Li,
    • Anthony Squire,
    • Tobias Tertel,
    • Bernd Giebel,
    • Peter Ludewig,
    • Philippa Spangenberg,
    • Jianxu Chen,
    • Axel Mosig,
    • Matthias Gunzer,
    • Dirk M. Hermann,
    Microvascular Network Remodeling in the Ischemic Mouse Brain Defined by Light Sheet Microscopy, Arteriosclerosis, Thrombosis, and Vascular Biology, 44, 4, (915-929), (2024)./doi/10.1161/ATVBAHA.123.320339
    Abstract
    • Gustavo H. Oliveira-Paula,
    • Sophia Liu,
    • Alishba Maira,
    • Gaia Ressa,
    • Graziele C. Ferreira,
    • Amado Quintar,
    • Smitha Jayakumar,
    • Vanessa Almonte,
    • Dippal Parikh,
    • Tomas Valenta,
    • Konrad Basler,
    • Timothy Hla,
    • Dario F. Riascos-Bernal,
    • Nicholas E. S. Sibinga,
    The β-catenin C terminus links Wnt and sphingosine-1-phosphate signaling pathways to promote vascular remodeling and atherosclerosis, Science Advances, 10, 11, (2024).https://doi.org/10.1126/sciadv.adg9278
    Crossref
    • Ilaria Del Gaudio,
    • Anja Nitzsche,
    • Kevin Boyé,
    • Philippe Bonnin,
    • Mathilde Poulet,
    • Toan Q Nguyen,
    • Ludovic Couty,
    • Hoa T T Ha,
    • Dat T Nguyen,
    • Amaury Cazenave-Gassiot,
    • Khaoula Ben Alaya,
    • Patrice Thérond,
    • Jerold Chun,
    • Markus R Wenk,
    • Richard L Proia,
    • Daniel Henrion,
    • Long N Nguyen,
    • Anne Eichmann,
    • Eric Camerer,
    Zonation and ligand and dose dependence of sphingosine 1-phosphate receptor-1 signalling in blood and lymphatic vasculature, Cardiovascular Research, 120, 14, (1794-1810), (2024).https://doi.org/10.1093/cvr/cvae168
    Crossref
    • Juan Yang,
    • Ziyu Li,
    • Xinyi Deng,
    • Mengru Li,
    • Bin Li,
    • Rebecca Caroline Thuku,
    • Qian Chen,
    • Xiang Sun,
    • Qiumin Lu,
    • Mingqian Fang,
    Kallikrein inhibitor derived from immunoglobulin heavy chain junction region possesses anti-thromboinflammation potential, Pharmacological Research, 209, (107460), (2024).https://doi.org/10.1016/j.phrs.2024.107460
    Crossref
  11. See more

View Options

View options

PDF and All Supplements

Download PDF and All Supplements

PDF/EPUB

View PDF/EPUB
Login options

Check if you have access through your login credentials or your institution to get full access on this article.

Personal login Institutional Login
Save for later Item saved, go to cart
Purchase Options

Purchase this article to access the full text.

Purchase access to this article for 24 hours

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke
Circulation Research
  • Vol. 128
  • No. 3

Purchase access to this journal for 24 hours

Circulation Research
  • Vol. 128
  • No. 3
Restore your content access

Enter your email address to restore your content access:

Note: This functionality works only for purchases done as a guest. If you already have an account, log in to access the content to which you are entitled.

Figures

Tables

Media

Share

Share

Share article link

Share

View full text|Download PDF

Comment Response