Sphingosine-1-Phosphate Receptor-1 Selective Agonist Enhances Collateral Growth and Protects against Subsequent Stroke

Abstract

Background and purpose: Collateral growth after acute occlusion of an intracranial artery is triggered by increasing shear stress in preexisting collateral pathways. Recently, sphingosine-1-phosphate receptor-1 (S1PR1) on endothelial cells was reported to be essential in sensing fluid shear stress. Here, we evaluated the expression of S1PR1 in the hypoperfused mouse brain and investigated the effect of a selective S1PR1 agonist on leptomeningeal collateral growth and subsequent ischemic damage after focal ischemia.

Methods: In C57Bl/6 mice (n = 133) subjected to unilateral common carotid occlusion (CCAO) and sham surgery. The first series examined the time course of collateral growth, cell proliferation, and S1PR1 expression in the leptomeningeal arteries after CCAO. The second series examined the relationship between pharmacological regulation of S1PR1 and collateral growth of leptomeningeal anastomoses. Animals were randomly assigned to one of the following groups: LtCCAO and daily intraperitoneal (i.p.) injection for 7 days of an S1PR1 selective agonist (SEW2871, 5 mg/kg/day); sham surgery and daily i.p. injection for 7 days of SEW2871 after surgery; LtCCAO and daily i.p. injection for 7 days of SEW2871 and an S1PR1 inverse agonist (VPC23019, 0.5 mg/kg); LtCCAO and daily i.p. injection of DMSO for 7 days after surgery; and sham surgery and daily i.p. injection of DMSO for 7 days. Leptomeningeal anastomoses were visualized 14 days after LtCCAO by latex perfusion method, and a set of animals underwent subsequent permanent middle cerebral artery occlusion (pMCAO) 7 days after the treatment termination. Neurological functions 1 hour, 1, 4, and 7 days and infarction volume 7 days after pMCAO were evaluated.

Results: In parallel with the increase in S1PR1 mRNA levels, S1PR1 expression colocalized with endothelial cell markers in the leptomeningeal arteries, increased markedly on the side of the CCAO, and peaked 7 days after CCAO. Mitotic cell numbers in the leptomeningeal arteries increased after CCAO. Administration of the S1PR1 selective agonist significantly increased cerebral blood flow (CBF) and the diameter of leptomeningeal collateral vessels (42.9 ± 2.6 μm) compared with the controls (27.6 ± 5.7 μm; P < 0.01). S1PR1 inverse agonist administration diminished the effect of the S1PR1 agonist (P < 0.001). After pMCAO, S1PR1 agonist pretreated animals showed significantly smaller infarct volume (17.5% ± 4.0% vs. 7.7% ± 4.0%, P < 0.01) and better functional recovery than vehicle-treated controls.

Conclusions: These results suggest that S1PR1 is one of the principal regulators of leptomeningeal collateral recruitment at the site of increased shear stress and provide evidence that an S1PR1 selective agonist has a role in promoting collateral growth and preventing of ischemic damage and neurological dysfunction after subsequent stroke in patients with intracranial major artery stenosis or occlusion.

Fig 1. Ischemic surgery and treatment schedule.

In the protocol I, animals were euthanized 1, 4, 7, or 14 days after surgery (A). In protocol II (B) and III (C). SEW, SEW+VPC, or control vehicle (DMSO) were administered intraperitoneally for 7 days after surgery.

Fig 2. Changes in CBF and leptomeningeal anastomosis after LtCCAO.

(A) Changes in CBF in the border zone between the middle cerebral artery (MCA) and anterior cerebral artery (ACA) after CCAO surgery (n = 7 for each group; ***P < 0.001 compared with sham; repeated-measures ANOVA followed by Tukey–Kramer post hoc test). (B) Representative images of superficial vessels on the ipsilateral hemisphere after LtCCAO, as assessed after latex perfusion (bars = 100 μm). Magnified images of the boxes in the upper panels are shown in the lower panels. Leptomeningeal artery between the ACA and MCA is indicated by arrows. The bar graphs indicates average diameter and average number of leptomeningeal anastomoses in the area between the ACA and MCA, as assessed after latex perfusion. CBF, cerebral blood flow; LtCCAO, left common cerebral artery occlusion.

Fig 3. Significant increase in the number of proliferating cells in the ipsilateral leptomeningeal arteries after LtCCAO.

Representative images of α-smooth muscle actin staining (A) of the ipsilateral leptomeningeal artery 2 weeks after sham surgery or LtCCAO surgery (scale bars = 10 μm). Dot line indicates the surface of cerebral cortex. There was no significant increase in the average area of ipsilateral leptomeningeal arteries after LtCCAO (B). Representative images of Ki-67 staining of ipsilateral leptomeningeal arteries (bars = 10 μm). The average number of Ki-67-positive cells in the leptomeningeal arteries (per section) after LtCCAO surgery increased until 7 days and then decreased. (n = 6 for each group; **P < 0.01, ***P < 0.001 compared with sham-operated controls; one-way ANOVA followed by Tukey–Kramer post hoc test).

Fig 4. Increase in expression of sphingosine-1-phosphate receptor-1 (S1PR1) in ipsilateral leptomeningeal arteries after LtCCAO.

(A) Confocal immunofluorescence double-labeling images with anti-CD31 (green) and anti-S1PR1 (red) antibodies in the ipsilateral leptomeningeal arteries 7 and 14 days after LtCCAO and in the sham-operated control. The strong S1PR1 signals (red) were detected at 7 days, but weak at 14 days after LtCCAO (bars = 10 μm). (B) Average percentage of the CD31/S1PR1 double-positive area of the total CD31 positive area after LtCCAO increased until 7 days and then decreased. (n = 6 for each group; *P < 0.05, ***P < 0.001 compared with sham-operated controls; one-way ANOVA followed by Tukey–Kramer post hoc test). (C) Confocal immunofluorescence double-labeling images with anti-CD31 (green) and anti-S1PR1 (red) antibodies in the ipsilateral parenchyma 7 days after LtCCAO and in the sham-operated control (bars = 50 μm). (D) Measurement of S1PR1 mRNA levels in ipsilateral cortex of sham-operated and ltCCAO animals. (n = 2 for sham, 3 for each CCAO group; **P < 0.01, ***P < 0.001 compared with sham-operated controls; one-way ANOVA followed by Tukey–Kramer post hoc test).

Fig 5. SEW2871 treatment promotes leptomeningeal collateral growth under shear stress conditions.

(A) Changes in cerebral blood flow (CBF) in the border zone between the anterior cerebral artery (ACA) and middle cerebral artery (MCA) in each group (n = 11 for vehicle, 11 for SEW, and 8 for SEW+VPC; ***P < 0.001 compared with vehicle, ^††† P < 0.001 compared with SEW+VPC group; repeated-measures ANOVA followed by Tukey–Kramer post hoc test.) (B) Representative images of superficial vessels on ipsilateral hemisphere after left common carotid artery occlusion (LtCCAO), as assessed after latex perfusion. Magnified images of boxes in the upper panels are shown in the lower panels. Arrows indicate leptomeningeal anastomoses between the ACA and MCA (bars = 100 μm). (C) Average diameters and average numbers of leptomeningeal anastomoses between the ACA and MCA, as assessed after latex perfusion. (n = 7 for vehicle, 7 for SEW, 6 for SEW without CCAO, and 4 for SEW+VPC ***P < 0.001 compared with vehicle group; one-way ANOVA followed by Tukey–Kramer post hoc test). SEW, sphingosine-1-phosphate receptor-1 (S1PR1) selective agonist; VPC, S1PR1 inverse agonist.

Fig 6. Total infarction volume reduction and improvement in neurological dysfunction with SEW treatment before pMCAO.

(A) Cresyl violet staining of the brain after pMCAO in the sham group, vehicle group, and SEW group is represented. Bar graph indicates total infarct volumes measured in eight coronal sections from the sham group, vehicle group, and SEW group 1 week after pMCAO. (B) Cerebral blood flow in the border zone 24 h after pMCAO (left). Average areas of ipsilateral leptomeningeal arteries in the sham, vehicle, and SEW groups (right). (C) The neurological deficit score 24 h after pMCAO (left) was less severe in the SEW group than in the vehicle or sham group (n = 5 for sham, 6 for vehicle, and 6 for SEW; ^† P < 0.05, ^†† P < 0.01, ^††† P < 0.001 compared with sham group, *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle group by one-way ANOVA followed by Tukey–Kramer post hoc test). As assessed by the elevated body swing test (right), animals displayed more frequent turns toward the contralateral side (right) after pMCAO. Animals in the SEW group showed significantly improved recovery of right-biased body swing rate compared with those in the sham and vehicle groups (n = 5 for sham, 6 for vehicle, and 6 for SEW; ^†† P < 0.01, ^† P < 0.05 compared with sham group, **P < 0.01, *P < 0.05 compared with vehicle group by repeated-measures ANOVA followed by Tukey–Kramer post hoc test). SEW, sphingosine-1-phosphate receptor-1 (S1PR1) selective agonist; pMCAO, permanent middle cerebral artery occlusion.