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Critical role of sphingosine-1-phosphate receptor-2 in the disruption of cerebrovascular integrity in experimental stroke.

Kim GS, Yang L, Zhang G, Zhao H, Selim M, McCullough LD, Kluk MJ, Sanchez T - Nat Commun (2015)

Bottom Line: In addition, inhibition of S1PR2 results in decreased matrix metalloproteinase (MMP)-9 activity in vivo and lower gelatinase activity in cerebral microvessels.S1PR2 immunopositivity is detected only in the ischemic microvessels of wild-type mice and in the cerebrovascular endothelium of human brain autopsy samples.In vitro, S1PR2 potently regulates the responses of the brain endothelium to ischaemic and inflammatory injury.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Emergency Medicine, the Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA [2] Department of Surgery, the Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.

ABSTRACT
The use and effectiveness of current stroke reperfusion therapies are limited by the complications of reperfusion injury, which include increased cerebrovascular permeability and haemorrhagic transformation. Sphingosine-1-phosphate (S1P) is emerging as a potent modulator of vascular integrity via its receptors (S1PR). By using genetic approaches and a S1PR2 antagonist (JTE013), here we show that S1PR2 plays a critical role in the induction of cerebrovascular permeability, development of intracerebral haemorrhage and neurovascular injury in experimental stroke. In addition, inhibition of S1PR2 results in decreased matrix metalloproteinase (MMP)-9 activity in vivo and lower gelatinase activity in cerebral microvessels. S1PR2 immunopositivity is detected only in the ischemic microvessels of wild-type mice and in the cerebrovascular endothelium of human brain autopsy samples. In vitro, S1PR2 potently regulates the responses of the brain endothelium to ischaemic and inflammatory injury. Therapeutic targeting of this novel pathway could have important translational relevance to stroke patients.

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Role of S1PR2 in the responses of neurons and glial cells to injuryMouse primary neurons (A–C) and mouse primary mixed glial cells (D, E) were isolated and cultured as described in the method section. A) Mouse primary neurons were subjected to 4 hours of OGD. 6 or 24 hours after in vitro reperfusion RNA was isolated and S1PR2 mRNA copy number was calculated by RT-qPCR analysis and normalized by 106 copies of 18s RNA. Fold induction vs control (C, normoxia, normoglycemia) is plotted. B) Mouse primary neurons were subjected to 4 hours of OGD. Upon in vitro reperfusion, cells were treated with vehicle (V), α-PBN (1mM) or JTE013 (1μM) for 24 hours. Neuronal cell death was assessed by measuring LDH activity in the supernatants. C) Primary neurons were treated with H2O2 (30μM) for 24 hours in the presence or absence of 1mM α-PBN or 1μM JTE 013 (JTE) and LDH activity was measured. Control: no H2O2. V: vehicle. D, E) Mouse primary mixed glial cells were activated with 5ng/mL TNF-α to mimic the proinflammatory environment of I/R injury. 24 hours later, S1PR2 (D) and IL-1β (E) mRNA levels were assessed by RT-qPCR analysis. F) Mouse primary mixed glial cells were activated with 5ng/mL TNF-α in the presence or absence (vehicle) of JTE013 at the doses indicated (μM). MMP-9 and MMP-2 activity in the conditioned media was assessed by gelatin zymography. Values are mean ± SEM. n=3 from 3 independent experiments. A representative zymography is shown. * P≤0.05 TNF-α vs control, and when indicated, TNF-α vs TNF-α+JTE013-treated (one-way ANOVA followed by Newman-Keuls). St: active MMP-9 standard. A–E) Fold induction vs control is plotted.
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Figure 7: Role of S1PR2 in the responses of neurons and glial cells to injuryMouse primary neurons (A–C) and mouse primary mixed glial cells (D, E) were isolated and cultured as described in the method section. A) Mouse primary neurons were subjected to 4 hours of OGD. 6 or 24 hours after in vitro reperfusion RNA was isolated and S1PR2 mRNA copy number was calculated by RT-qPCR analysis and normalized by 106 copies of 18s RNA. Fold induction vs control (C, normoxia, normoglycemia) is plotted. B) Mouse primary neurons were subjected to 4 hours of OGD. Upon in vitro reperfusion, cells were treated with vehicle (V), α-PBN (1mM) or JTE013 (1μM) for 24 hours. Neuronal cell death was assessed by measuring LDH activity in the supernatants. C) Primary neurons were treated with H2O2 (30μM) for 24 hours in the presence or absence of 1mM α-PBN or 1μM JTE 013 (JTE) and LDH activity was measured. Control: no H2O2. V: vehicle. D, E) Mouse primary mixed glial cells were activated with 5ng/mL TNF-α to mimic the proinflammatory environment of I/R injury. 24 hours later, S1PR2 (D) and IL-1β (E) mRNA levels were assessed by RT-qPCR analysis. F) Mouse primary mixed glial cells were activated with 5ng/mL TNF-α in the presence or absence (vehicle) of JTE013 at the doses indicated (μM). MMP-9 and MMP-2 activity in the conditioned media was assessed by gelatin zymography. Values are mean ± SEM. n=3 from 3 independent experiments. A representative zymography is shown. * P≤0.05 TNF-α vs control, and when indicated, TNF-α vs TNF-α+JTE013-treated (one-way ANOVA followed by Newman-Keuls). St: active MMP-9 standard. A–E) Fold induction vs control is plotted.

Mentions: In order to assess the role of S1PR2 in the regulation of neuronal and glial responses to ischemic and inflammatory injury, we isolated and cultured mouse cortical primary neurons from embryos (E 17.5) and mixed glial cells from pups (post natal day 2). In vitro I/R injury (4 hours of OGD followed by in vitro reperfusion) did not significantly change the levels of S1PR2 mRNA in mouse cortical primary neurons (Figure 7A). In addition, blockade of S1PR2 signaling by JTE013 did not inhibit neuronal cell death induced by in vitro I/R injury, assessed by LDH activity in neuronal supernatants (Figure 7B), while α-phenyl-tert-butyl nitrone α-(PBN), a neuroprotective spin trap agent that reacts with and trap free radicals 35, did inhibit. Similarly, neuronal cell death induced by H2O2 was not affected by JTE013 but it was inhibited by α-PBN (Figure 7C). In mouse primary mixed glial cells, activation with TNF-α did not significantly increase the levels of S1PR2 mRNA (Figure 7D) while it significantly induced the expression of other pro-inflammatory molecules, such as interleukin 1β (Figure 7E). In addition, we found that JTE013 did not affect the inflammatory responses of mouse primary mixed glial cells. As shown in Figure 7F, the induction of MMP-9 activity by TNF-α in glial cell supernatants was not inhibited by the S1PR2 antagonist, JTE013. These data indicate that S1PR2 is not critical for the regulation of neuronal and glial responses to ischemic and inflammatory injury in vitro. Altogether our in vitro data with brain endothelial cells, neurons and glial cells indicate that S1PR2 plays a critical role in the responses of brain endothelial cells to I/R injury.


Critical role of sphingosine-1-phosphate receptor-2 in the disruption of cerebrovascular integrity in experimental stroke.

Kim GS, Yang L, Zhang G, Zhao H, Selim M, McCullough LD, Kluk MJ, Sanchez T - Nat Commun (2015)

Role of S1PR2 in the responses of neurons and glial cells to injuryMouse primary neurons (A–C) and mouse primary mixed glial cells (D, E) were isolated and cultured as described in the method section. A) Mouse primary neurons were subjected to 4 hours of OGD. 6 or 24 hours after in vitro reperfusion RNA was isolated and S1PR2 mRNA copy number was calculated by RT-qPCR analysis and normalized by 106 copies of 18s RNA. Fold induction vs control (C, normoxia, normoglycemia) is plotted. B) Mouse primary neurons were subjected to 4 hours of OGD. Upon in vitro reperfusion, cells were treated with vehicle (V), α-PBN (1mM) or JTE013 (1μM) for 24 hours. Neuronal cell death was assessed by measuring LDH activity in the supernatants. C) Primary neurons were treated with H2O2 (30μM) for 24 hours in the presence or absence of 1mM α-PBN or 1μM JTE 013 (JTE) and LDH activity was measured. Control: no H2O2. V: vehicle. D, E) Mouse primary mixed glial cells were activated with 5ng/mL TNF-α to mimic the proinflammatory environment of I/R injury. 24 hours later, S1PR2 (D) and IL-1β (E) mRNA levels were assessed by RT-qPCR analysis. F) Mouse primary mixed glial cells were activated with 5ng/mL TNF-α in the presence or absence (vehicle) of JTE013 at the doses indicated (μM). MMP-9 and MMP-2 activity in the conditioned media was assessed by gelatin zymography. Values are mean ± SEM. n=3 from 3 independent experiments. A representative zymography is shown. * P≤0.05 TNF-α vs control, and when indicated, TNF-α vs TNF-α+JTE013-treated (one-way ANOVA followed by Newman-Keuls). St: active MMP-9 standard. A–E) Fold induction vs control is plotted.
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Figure 7: Role of S1PR2 in the responses of neurons and glial cells to injuryMouse primary neurons (A–C) and mouse primary mixed glial cells (D, E) were isolated and cultured as described in the method section. A) Mouse primary neurons were subjected to 4 hours of OGD. 6 or 24 hours after in vitro reperfusion RNA was isolated and S1PR2 mRNA copy number was calculated by RT-qPCR analysis and normalized by 106 copies of 18s RNA. Fold induction vs control (C, normoxia, normoglycemia) is plotted. B) Mouse primary neurons were subjected to 4 hours of OGD. Upon in vitro reperfusion, cells were treated with vehicle (V), α-PBN (1mM) or JTE013 (1μM) for 24 hours. Neuronal cell death was assessed by measuring LDH activity in the supernatants. C) Primary neurons were treated with H2O2 (30μM) for 24 hours in the presence or absence of 1mM α-PBN or 1μM JTE 013 (JTE) and LDH activity was measured. Control: no H2O2. V: vehicle. D, E) Mouse primary mixed glial cells were activated with 5ng/mL TNF-α to mimic the proinflammatory environment of I/R injury. 24 hours later, S1PR2 (D) and IL-1β (E) mRNA levels were assessed by RT-qPCR analysis. F) Mouse primary mixed glial cells were activated with 5ng/mL TNF-α in the presence or absence (vehicle) of JTE013 at the doses indicated (μM). MMP-9 and MMP-2 activity in the conditioned media was assessed by gelatin zymography. Values are mean ± SEM. n=3 from 3 independent experiments. A representative zymography is shown. * P≤0.05 TNF-α vs control, and when indicated, TNF-α vs TNF-α+JTE013-treated (one-way ANOVA followed by Newman-Keuls). St: active MMP-9 standard. A–E) Fold induction vs control is plotted.
Mentions: In order to assess the role of S1PR2 in the regulation of neuronal and glial responses to ischemic and inflammatory injury, we isolated and cultured mouse cortical primary neurons from embryos (E 17.5) and mixed glial cells from pups (post natal day 2). In vitro I/R injury (4 hours of OGD followed by in vitro reperfusion) did not significantly change the levels of S1PR2 mRNA in mouse cortical primary neurons (Figure 7A). In addition, blockade of S1PR2 signaling by JTE013 did not inhibit neuronal cell death induced by in vitro I/R injury, assessed by LDH activity in neuronal supernatants (Figure 7B), while α-phenyl-tert-butyl nitrone α-(PBN), a neuroprotective spin trap agent that reacts with and trap free radicals 35, did inhibit. Similarly, neuronal cell death induced by H2O2 was not affected by JTE013 but it was inhibited by α-PBN (Figure 7C). In mouse primary mixed glial cells, activation with TNF-α did not significantly increase the levels of S1PR2 mRNA (Figure 7D) while it significantly induced the expression of other pro-inflammatory molecules, such as interleukin 1β (Figure 7E). In addition, we found that JTE013 did not affect the inflammatory responses of mouse primary mixed glial cells. As shown in Figure 7F, the induction of MMP-9 activity by TNF-α in glial cell supernatants was not inhibited by the S1PR2 antagonist, JTE013. These data indicate that S1PR2 is not critical for the regulation of neuronal and glial responses to ischemic and inflammatory injury in vitro. Altogether our in vitro data with brain endothelial cells, neurons and glial cells indicate that S1PR2 plays a critical role in the responses of brain endothelial cells to I/R injury.

Bottom Line: In addition, inhibition of S1PR2 results in decreased matrix metalloproteinase (MMP)-9 activity in vivo and lower gelatinase activity in cerebral microvessels.S1PR2 immunopositivity is detected only in the ischemic microvessels of wild-type mice and in the cerebrovascular endothelium of human brain autopsy samples.In vitro, S1PR2 potently regulates the responses of the brain endothelium to ischaemic and inflammatory injury.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Emergency Medicine, the Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA [2] Department of Surgery, the Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.

ABSTRACT
The use and effectiveness of current stroke reperfusion therapies are limited by the complications of reperfusion injury, which include increased cerebrovascular permeability and haemorrhagic transformation. Sphingosine-1-phosphate (S1P) is emerging as a potent modulator of vascular integrity via its receptors (S1PR). By using genetic approaches and a S1PR2 antagonist (JTE013), here we show that S1PR2 plays a critical role in the induction of cerebrovascular permeability, development of intracerebral haemorrhage and neurovascular injury in experimental stroke. In addition, inhibition of S1PR2 results in decreased matrix metalloproteinase (MMP)-9 activity in vivo and lower gelatinase activity in cerebral microvessels. S1PR2 immunopositivity is detected only in the ischemic microvessels of wild-type mice and in the cerebrovascular endothelium of human brain autopsy samples. In vitro, S1PR2 potently regulates the responses of the brain endothelium to ischaemic and inflammatory injury. Therapeutic targeting of this novel pathway could have important translational relevance to stroke patients.

Show MeSH
Related in: MedlinePlus