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Protein S controls hypoxic/ischemic blood-brain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor.

ABSTRACT: The anticoagulant factor protein S (PS) has direct cellular activities. Lack of PS in mice causes lethal coagulopathy, ischemic/thrombotic injuries, vascular dysgenesis, and blood-brain barrier (BBB) disruption with intracerebral hemorrhages. Thus, we hypothesized that PS maintains and/or enhances the BBB integrity. Using a BBB model with human brain endothelial cells, we show PS inhibits time- and dose-dependently (half maximal effective concentration [EC(50)] = 27 +/- 3 nM) oxygen/glucose deprivation-induced BBB breakdown, as demonstrated by measurements of the transmonolayer electrical resistance, permeability of endothelial monolayers to dextran (40 kDa), and rearrangement of F-actin toward the cortical cytoskeletal ring. Using Tyro-3, Axl, and Mer (TAM) receptor, tyrosine kinase silencing through RNA interference, specific N-terminus-blocking antibodies, Tyro3 phosphorylation, and Tyro3-, Axl- and Mer-deficient mouse brain endothelial cells, we show that Tyro3 mediates PS vasculoprotection. After Tyro3 ligation, PS activated sphingosine 1-phosphate receptor (S1P(1)), resulting in Rac1-dependent BBB protection. Using 2-photon in vivo imaging, we show that PS blocks postischemic BBB disruption in Tyro3(+/+), Axl(-/-), and Mer(-/-) mice, but not in Tyro3(-/-) mice or Tyro3(+/+) mice receiving low-dose W146, a S1P(1)-specific antagonist. Our findings indicate that PS protects the BBB integrity via Tyro3 and S1P(1), suggesting potentially novel treatments for neurovascular dysfunction resulting from hypoxic/ischemic BBB damage.

SUBMITTER: Zhu D

PROVIDER: S-EPMC2890172 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Protein S controls hypoxic/ischemic blood-brain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor.

Zhu Donghui D Wang Yaoming Y Singh Itender I Bell Robert D RD Deane Rashid R Zhong Zhihui Z Sagare Abhay A Winkler Ethan A EA Zlokovic Berislav V BV

Blood 20100326 23

The anticoagulant factor protein S (PS) has direct cellular activities. Lack of PS in mice causes lethal coagulopathy, ischemic/thrombotic injuries, vascular dysgenesis, and blood-brain barrier (BBB) disruption with intracerebral hemorrhages. Thus, we hypothesized that PS maintains and/or enhances the BBB integrity. Using a BBB model with human brain endothelial cells, we show PS inhibits time- and dose-dependently (half maximal effective concentration [EC(50)] = 27 +/- 3 nM) oxygen/glucose depr ...[more]

PMID: 20348395

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Project description:Vascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms. To experimentally define the effects of FTY720 regioisomers on lung endothelial cell barrier regulation. Specific barrier-regulatory receptor and kinase inhibitors were utilized to probe signaling mechanisms involved in FTY720 regioisomer-mediated human lung endothelial cell barrier responses (trans-endothelial electrical resistance, TER). Docking simulations with the S1P1 receptor were performed to further evaluate FTY720 regioisomer signaling. FTY720 regioisomers produced potent endothelial cell barrier disruption reflected by declines in TER alterations. Pharmacologic inhibition of Gi-coupled S1P receptors (S1P1, S1P2, S1P3) failed to alter FTY720 regioisomer-mediated barrier disruption; findings that were corroborated by docking simulations demonstrating FTY720 regiosomers were repelled from S1P1 docking, in contrast to strong S1P1 binding elicited by S1P. Inhibition of either the barrier-disrupting PAR-1 receptor, the VEGF receptor, Rho-kinase, MAPK, NFkB, or PI3K failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. While FTY720 regioisomers significantly increased protein phosphatase 2 (PP2A) activity, PP2A inhibitors failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. Together, these results imply a vexing model of pulmonary vascular barrier dysregulation in response to FTY720-related compounds and highlight the need for further insights into mechanisms of vascular integrity required to promote the development of novel therapeutic tools to prevent or reverse the pulmonary vascular leak central to ARDS outcomes.

Project description:BackgroundTraumatic brain injury (TBI) provokes secondary pathological damage, such as damage to the blood-brain barrier (BBB), ischaemia and inflammation. Major facilitator superfamily domain-containing 2a (Mfsd2a) has been demonstrated to be critical in limiting the increase in BBB vesicle transcytosis following brain injury. Recent studies suggest that a novel and selective modulator of the sphingosine-1-phosphate receptor 1 (S1P1), CYM-5442, maintains the integrity of the BBB by restricting vesicle transcytosis during acute ischaemic stroke. In the current study, we investigated whether CYM-5442, evaluated in a short-term study, could protect the brains of mice with acute-stage TBI by reversing the increase in vesicle transport due to reduced Mfsd2a expression after TBI.MethodsWe used the well-characterized model of TBI caused by controlled cortical impact. CYM-5442 (0.3, 1, 3 mg/kg) was intraperitoneally injected 30 min after surgery for 7 consecutive days. To investigate the effect of CYM-5442 on vesicle transcytosis, we downregulated and upregulated Mfsd2a expression using a specific AAV prior to evaluation of the TBI model. MRI scanning, cerebral blood flow, circulating blood counts, ELISA, TEM, WB, and immunostaining evaluations were performed after brain injury.ResultsCYM-5442 significantly attenuated neurological deficits and reduced brain oedema in TBI mice. CYM-5442 transiently suppressed lymphocyte trafficking but did not induce persistent lymphocytopenia. After TBI, the levels of Mfsd2a were decreased significantly, while the levels of CAV-1 and albumin were increased. In addition, Mfsd2a deficiency caused inadequate sphingosine-1-phosphate (S1P) transport in the brain parenchyma, and the regulation of BBB permeability by Mfsd2a after TBI was shown to be related to changes in vesicle transcytosis. Downregulation of Mfsd2a in mice markedly increased the BBB permeability, neurological deficit scores, and brain water contents after TBI. Intervention with CYM-5442 after TBI protected the BBB by significantly reducing the vesicle transcytosis of cerebrovascular endothelial cells.ConclusionIn addition to transiently suppressing lymphocytes, CYM-5442 alleviated the neurological deficits, cerebral edema and protective BBB permeability in TBI mice by reducing the vesicle transcytosis of cerebrovascular endothelial cells.

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Protein S controls hypoxic/ischemic blood-brain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor.

Publications

Protein S controls hypoxic/ischemic blood-brain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor.

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