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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

Effects of AKB-9785 treatment in vitro. a MBMECs pre-treated with hAng-2 increased Tie2/Akt activation upon AKB-9785 treatment (n = 3). b bEnd5 Western blots revealed Tie2/Akt activation, but a decrease (non-significant) in pVE-cadherin by AKB-9785 (n = 3). c MBMECs pre-treated with recombinant hAng-2 [500 ng/ml] followed by AKB-9785 [1 µM] treatment revealed increased TEER values. d The in vitro permeability tracer experiment with a 3 kD Texas Red-dextran showed a decrease in permeability with AKB-9785 + hAng-2 compared to hAng-2 treatment alone. This difference was not detected in the presence of filipin. e The phosphorylation of Akt in Western Blot analysis could only be identified in MBMECs, but not pericytes (n = 3)
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Fig8: Effects of AKB-9785 treatment in vitro. a MBMECs pre-treated with hAng-2 increased Tie2/Akt activation upon AKB-9785 treatment (n = 3). b bEnd5 Western blots revealed Tie2/Akt activation, but a decrease (non-significant) in pVE-cadherin by AKB-9785 (n = 3). c MBMECs pre-treated with recombinant hAng-2 [500 ng/ml] followed by AKB-9785 [1 µM] treatment revealed increased TEER values. d The in vitro permeability tracer experiment with a 3 kD Texas Red-dextran showed a decrease in permeability with AKB-9785 + hAng-2 compared to hAng-2 treatment alone. This difference was not detected in the presence of filipin. e The phosphorylation of Akt in Western Blot analysis could only be identified in MBMECs, but not pericytes (n = 3)

Mentions: We had previously observed increased Ang-2 levels in WT rodents subjected to MCAO [7] and thus sought to investigate the rescue of stroke size/permeability in WT mice by interference with Ang-2/Tie2 signaling. To this end, we employed the more clinically relevant transient MCAO (tMCAO) model in WT mice and aimed a rescue with AKB-9785 (gift from Aerpio Therapeutics), a drug targeting Tie2 signaling by blocking the activity of the vascular endothelial protein tyrosine phosphatase (VE-PTP) that is associated with Tie2 and VE-cadherin [54]. In vitro, MBMECs/bEnd5 pre-treated with hAng-2 followed by AKB-9785 treatment showed an increase in pTie2 and pAkt, but not in pVE-cadherin (Fig. 8a, b), associated with decreased junctional permeability as shown by increased TEER of MBMECs after 24 and 48 h (Fig. 8c) as well as decreased capacitance (Ccl) values (Suppl. Fig. 4b). The permeability effect of hAng-2 decreased in combination with AKB-9785 (Fig. 8c, d). Upon filipin treatment, a compound blocking the formation of caveolae, no differences in 3 kD dextran tracer between control, hAng-2 and hAng-2 plus AKB-9785 could be detected anymore (Fig. 8d). These data support our hypothesis that hAng-2 causes permeability partly by transcellular vesicular permeability and that AKB-9785 is able to prevent this effect. In the 67 kD BSA-Alexa 647 tracer, this effect was abolished, suggesting that Ang-2 induced BBB permeability primarily facilitates transition of smaller molecules (Suppl. Fig. 4c). The effect of AKB-9785 is endothelial specific, as phosphorylation of Akt could neither be detected in pericytes (Fig. 8e) nor in astrocytes (Suppl. Fig. 4d). WT tMCAO mice treated with AKB-9785 displayed reduced infarcts (Fig. 9a) and permeability to IgG (Fig. 9c) with a post-stroke neurological improvement 72 h after occlusion (Fig. 9b). The AKB-9785-treated hemispheres from mice subjected to tMCAO showed increased pTie2/pAkt activity (Fig. 9d, e). This finding is indicative of a restoration of the Tie2 signaling as pTie2 staining in AKB-9785-treated stroke brains appeared almost similar to pTie2 staining in normal brain vessels (unaffected hemispheres; Suppl. Fig. 5, compare to Fig. 9e). We next ascertained whether the increased permeability upon stroke in Ang-2 GOF mice was a direct effect of Ang-2 or secondary to stroke that is associated with impaired BBB. To this end, we performed permeability analyses at 3 h post-stroke, a time point associated with minimal infarct sizes. The results revealed already increased extravasation of 3 kD TMR-dextran in Ang-2 GOF mice 3 h post-stroke suggesting that Ang-2-mediated permeability could contribute to edema formation and thus facilitate larger infarct size at later time points (Suppl. Fig. 4e). These results are further supported by the therapeutic rescue achieved by administration of AKB-9785, potentially reducing BBB leakage by VE-PTP-dependent activation of Tie2 signaling.Fig. 8


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)

Effects of AKB-9785 treatment in vitro. a MBMECs pre-treated with hAng-2 increased Tie2/Akt activation upon AKB-9785 treatment (n = 3). b bEnd5 Western blots revealed Tie2/Akt activation, but a decrease (non-significant) in pVE-cadherin by AKB-9785 (n = 3). c MBMECs pre-treated with recombinant hAng-2 [500 ng/ml] followed by AKB-9785 [1 µM] treatment revealed increased TEER values. d The in vitro permeability tracer experiment with a 3 kD Texas Red-dextran showed a decrease in permeability with AKB-9785 + hAng-2 compared to hAng-2 treatment alone. This difference was not detected in the presence of filipin. e The phosphorylation of Akt in Western Blot analysis could only be identified in MBMECs, but not pericytes (n = 3)
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Fig8: Effects of AKB-9785 treatment in vitro. a MBMECs pre-treated with hAng-2 increased Tie2/Akt activation upon AKB-9785 treatment (n = 3). b bEnd5 Western blots revealed Tie2/Akt activation, but a decrease (non-significant) in pVE-cadherin by AKB-9785 (n = 3). c MBMECs pre-treated with recombinant hAng-2 [500 ng/ml] followed by AKB-9785 [1 µM] treatment revealed increased TEER values. d The in vitro permeability tracer experiment with a 3 kD Texas Red-dextran showed a decrease in permeability with AKB-9785 + hAng-2 compared to hAng-2 treatment alone. This difference was not detected in the presence of filipin. e The phosphorylation of Akt in Western Blot analysis could only be identified in MBMECs, but not pericytes (n = 3)
Mentions: We had previously observed increased Ang-2 levels in WT rodents subjected to MCAO [7] and thus sought to investigate the rescue of stroke size/permeability in WT mice by interference with Ang-2/Tie2 signaling. To this end, we employed the more clinically relevant transient MCAO (tMCAO) model in WT mice and aimed a rescue with AKB-9785 (gift from Aerpio Therapeutics), a drug targeting Tie2 signaling by blocking the activity of the vascular endothelial protein tyrosine phosphatase (VE-PTP) that is associated with Tie2 and VE-cadherin [54]. In vitro, MBMECs/bEnd5 pre-treated with hAng-2 followed by AKB-9785 treatment showed an increase in pTie2 and pAkt, but not in pVE-cadherin (Fig. 8a, b), associated with decreased junctional permeability as shown by increased TEER of MBMECs after 24 and 48 h (Fig. 8c) as well as decreased capacitance (Ccl) values (Suppl. Fig. 4b). The permeability effect of hAng-2 decreased in combination with AKB-9785 (Fig. 8c, d). Upon filipin treatment, a compound blocking the formation of caveolae, no differences in 3 kD dextran tracer between control, hAng-2 and hAng-2 plus AKB-9785 could be detected anymore (Fig. 8d). These data support our hypothesis that hAng-2 causes permeability partly by transcellular vesicular permeability and that AKB-9785 is able to prevent this effect. In the 67 kD BSA-Alexa 647 tracer, this effect was abolished, suggesting that Ang-2 induced BBB permeability primarily facilitates transition of smaller molecules (Suppl. Fig. 4c). The effect of AKB-9785 is endothelial specific, as phosphorylation of Akt could neither be detected in pericytes (Fig. 8e) nor in astrocytes (Suppl. Fig. 4d). WT tMCAO mice treated with AKB-9785 displayed reduced infarcts (Fig. 9a) and permeability to IgG (Fig. 9c) with a post-stroke neurological improvement 72 h after occlusion (Fig. 9b). The AKB-9785-treated hemispheres from mice subjected to tMCAO showed increased pTie2/pAkt activity (Fig. 9d, e). This finding is indicative of a restoration of the Tie2 signaling as pTie2 staining in AKB-9785-treated stroke brains appeared almost similar to pTie2 staining in normal brain vessels (unaffected hemispheres; Suppl. Fig. 5, compare to Fig. 9e). We next ascertained whether the increased permeability upon stroke in Ang-2 GOF mice was a direct effect of Ang-2 or secondary to stroke that is associated with impaired BBB. To this end, we performed permeability analyses at 3 h post-stroke, a time point associated with minimal infarct sizes. The results revealed already increased extravasation of 3 kD TMR-dextran in Ang-2 GOF mice 3 h post-stroke suggesting that Ang-2-mediated permeability could contribute to edema formation and thus facilitate larger infarct size at later time points (Suppl. Fig. 4e). These results are further supported by the therapeutic rescue achieved by administration of AKB-9785, potentially reducing BBB leakage by VE-PTP-dependent activation of Tie2 signaling.Fig. 8

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