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Angiopoietin-2-induced blood-brain barrier compromise and increased stroke size are rescued by VE-PTP-dependent restoration of Tie2 signaling.

Gurnik S, Devraj K, Macas J, Yamaji M, Starke J, Scholz A, Sommer K, Di Tacchio M, Vutukuri R, Beck H, Mittelbronn M, Foerch C, Pfeilschifter W, Liebner S, Peters KG, Plate KH, Reiss Y - Acta Neuropathol. (2016)

Bottom Line: These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes.In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated.We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurology (Edinger Institute), Goethe University Medical School, Frankfurt, Germany.

ABSTRACT
The homeostasis of the central nervous system is maintained by the blood-brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.

No MeSH data available.


Related in: MedlinePlus

Ang-2 increases brain endothelial permeability in vitro. a MBMECs from WT and Ang-2 GOF brains were seeded on transwell inserts and transferred to a cellZscopeĀ® system to obtain continuous TEER values. The inset shows a transwell insert along with top and bottom electrodes typical of a cellZscopeĀ® device. The graph shows a representative experiment that indicates reduced TEER values in Ang-2 GOF MBMECs compared to WT that is sustained up to 48Ā h post-seeding when the monolayers reach confluency. b GOF MBMECs showed lower TEER than control cells at 24 and 48Ā h. c WT-MBMECs treated with hAng-2 resulted in reduced TEER. d hAng-2 treatment resulted in increased tracer flux across MBMECs (bā€“d, nĀ =Ā 3)
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Fig1: Ang-2 increases brain endothelial permeability in vitro. a MBMECs from WT and Ang-2 GOF brains were seeded on transwell inserts and transferred to a cellZscopeĀ® system to obtain continuous TEER values. The inset shows a transwell insert along with top and bottom electrodes typical of a cellZscopeĀ® device. The graph shows a representative experiment that indicates reduced TEER values in Ang-2 GOF MBMECs compared to WT that is sustained up to 48Ā h post-seeding when the monolayers reach confluency. b GOF MBMECs showed lower TEER than control cells at 24 and 48Ā h. c WT-MBMECs treated with hAng-2 resulted in reduced TEER. d hAng-2 treatment resulted in increased tracer flux across MBMECs (bā€“d, nĀ =Ā 3)

Mentions: The effect of Ang-2 on BBB permeability was first assessed by transendothelial electrical resistance (TEER) and capacitance (Ccl) measurements on the primary mouse brain endothelial cells (MBMECs) (Fig.Ā 1a). The TEER of MBMECs derived from Ang-2 GOF mice was lower compared to WT (Fig.Ā 1b), and Ccl was higher (Suppl. Fig.Ā 1a) indicating a barrier-opening effect of Ang-2. Similar results were obtained when WT-MBMECs were treated with recombinant Ang-2 (hAng-2, Fig.Ā 1c). The permeability to fluorescent dextrans (3, 20, and 70 kD) and Lucifer Yellow (LY 0.45 kD) [15] was also increased in TEER measurement by hAng-2 (Fig.Ā 1d), suggesting a direct effect of Ang-2 on brain EC permeability. In vivo, the permeability to LY and 3 kD dextrans (Texas Red and TMR) was significantly increased in Ang-2 GOF (Fig.Ā 2a, c), with no difference for Evans blue dye (~70Ā kD) potentially owing to a tight barrier in vivo (Fig.Ā 2b). The 0.45Ā kD LY and 3Ā kD dextran tracers were circulated for 4Ā min due to their rapid clearance from blood vessels at longer time points (Suppl. Fig.Ā 1b). Kidneys served as a control for tracer permeability that was not altered between GOF and WT (Suppl. Fig.Ā 1c). These results therefore demonstrate Ang-2-mediated brain endothelial permeability for low molecular weight solutes in vivo.Fig.Ā 1


Angiopoietin-2-induced blood-brain barrier compromise and increased stroke size are rescued by VE-PTP-dependent restoration of Tie2 signaling.

Gurnik S, Devraj K, Macas J, Yamaji M, Starke J, Scholz A, Sommer K, Di Tacchio M, Vutukuri R, Beck H, Mittelbronn M, Foerch C, Pfeilschifter W, Liebner S, Peters KG, Plate KH, Reiss Y - Acta Neuropathol. (2016)

Ang-2 increases brain endothelial permeability in vitro. a MBMECs from WT and Ang-2 GOF brains were seeded on transwell inserts and transferred to a cellZscopeĀ® system to obtain continuous TEER values. The inset shows a transwell insert along with top and bottom electrodes typical of a cellZscopeĀ® device. The graph shows a representative experiment that indicates reduced TEER values in Ang-2 GOF MBMECs compared to WT that is sustained up to 48Ā h post-seeding when the monolayers reach confluency. b GOF MBMECs showed lower TEER than control cells at 24 and 48Ā h. c WT-MBMECs treated with hAng-2 resulted in reduced TEER. d hAng-2 treatment resulted in increased tracer flux across MBMECs (bā€“d, nĀ =Ā 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835530&req=5

Fig1: Ang-2 increases brain endothelial permeability in vitro. a MBMECs from WT and Ang-2 GOF brains were seeded on transwell inserts and transferred to a cellZscopeĀ® system to obtain continuous TEER values. The inset shows a transwell insert along with top and bottom electrodes typical of a cellZscopeĀ® device. The graph shows a representative experiment that indicates reduced TEER values in Ang-2 GOF MBMECs compared to WT that is sustained up to 48Ā h post-seeding when the monolayers reach confluency. b GOF MBMECs showed lower TEER than control cells at 24 and 48Ā h. c WT-MBMECs treated with hAng-2 resulted in reduced TEER. d hAng-2 treatment resulted in increased tracer flux across MBMECs (bā€“d, nĀ =Ā 3)
Mentions: The effect of Ang-2 on BBB permeability was first assessed by transendothelial electrical resistance (TEER) and capacitance (Ccl) measurements on the primary mouse brain endothelial cells (MBMECs) (Fig.Ā 1a). The TEER of MBMECs derived from Ang-2 GOF mice was lower compared to WT (Fig.Ā 1b), and Ccl was higher (Suppl. Fig.Ā 1a) indicating a barrier-opening effect of Ang-2. Similar results were obtained when WT-MBMECs were treated with recombinant Ang-2 (hAng-2, Fig.Ā 1c). The permeability to fluorescent dextrans (3, 20, and 70 kD) and Lucifer Yellow (LY 0.45 kD) [15] was also increased in TEER measurement by hAng-2 (Fig.Ā 1d), suggesting a direct effect of Ang-2 on brain EC permeability. In vivo, the permeability to LY and 3 kD dextrans (Texas Red and TMR) was significantly increased in Ang-2 GOF (Fig.Ā 2a, c), with no difference for Evans blue dye (~70Ā kD) potentially owing to a tight barrier in vivo (Fig.Ā 2b). The 0.45Ā kD LY and 3Ā kD dextran tracers were circulated for 4Ā min due to their rapid clearance from blood vessels at longer time points (Suppl. Fig.Ā 1b). Kidneys served as a control for tracer permeability that was not altered between GOF and WT (Suppl. Fig.Ā 1c). These results therefore demonstrate Ang-2-mediated brain endothelial permeability for low molecular weight solutes in vivo.Fig.Ā 1

Bottom Line: These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes.In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated.We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurology (Edinger Institute), Goethe University Medical School, Frankfurt, Germany.

ABSTRACT
The homeostasis of the central nervous system is maintained by the blood-brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.

No MeSH data available.


Related in: MedlinePlus