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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

Detection of the glycocalyx and plasma tracers by EM analysis: a lanthanum nitrate was used to detect the glycocalyx (black arrows) in mouse brain vessels. Glycocalyx was decreased/disrupted from ~300 to ~100 nm in GOF mice. b HRP was intravenously injected into WT and Ang-2 GOF mice and circulated for 30 min. Representative images of Ang-2 GOF mice revealed HRP-vesicles (black arrows) with affected endothelial junctions (red arrows). Quantitative analysis indicated more HRP-positive vesicles in Ang-2 GOF mice (20 vessels analyzed; n = 3; scale bar sizes are indicated in the images). c MBMV Western blots showed decreased VE-cadherin and claudin-5 in GOF, whereas caveolin-1 was upregulated (n = 3)
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Fig5: Detection of the glycocalyx and plasma tracers by EM analysis: a lanthanum nitrate was used to detect the glycocalyx (black arrows) in mouse brain vessels. Glycocalyx was decreased/disrupted from ~300 to ~100 nm in GOF mice. b HRP was intravenously injected into WT and Ang-2 GOF mice and circulated for 30 min. Representative images of Ang-2 GOF mice revealed HRP-vesicles (black arrows) with affected endothelial junctions (red arrows). Quantitative analysis indicated more HRP-positive vesicles in Ang-2 GOF mice (20 vessels analyzed; n = 3; scale bar sizes are indicated in the images). c MBMV Western blots showed decreased VE-cadherin and claudin-5 in GOF, whereas caveolin-1 was upregulated (n = 3)

Mentions: We next elaborated the molecular components of Ang-2-mediated permeability by immunohistochemistry (IHC), electron microscopy (EM) and Western blots. Quantitative RT-PCR analysis confirmed an overexpression of hAng-2 in the isolated brain microvessels of Ang-2 GOF mice, whereas Tie2 expression levels were not altered (Suppl. Fig. 1d). Furthermore, we did not observe an increase in Ang-1 levels (Suppl. Fig. 1e, f) in these mice, suggesting an absence of its compensatory effects. IHC from cryosections (10 µm) and from vibratome sections (50 µm) revealed a decrease in pericytes in GOF (Fig. 3a, b; Suppl. video) as previously observed in the periphery [53]. In Fig. 3a, we set the WT pericyte coverage to 100 %; however, the absolute pericyte coverage of the vessels is only about 20–25 % as obtained from vibratome data analysis which is reduced to approximately 12–15 % in the GOF mice. Also by EM analysis, we observed decreased pericyte coverage with increased numbers of degenerating pericytes in Ang-2 GOF mice (Fig. 4a–c; Suppl. Table 1). EM analysis also showed increased caveolae-like vesicles (Fig. 4a, e) that are partly responsible for peripheral endothelial permeability. The analysis also revealed decreased complexity of the inter-endothelial junctions in GOF, with frequent gaps (Fig. 4a, d; Suppl. Fig. 2a; Suppl. Table 2), indicating increased paracellular permeability. Furthermore, the glycocalyx thickness was considerably disrupted/decreased (~300–~100 nm, Fig. 5a; Suppl. Fig. 2b). This finding implies increased permeability induced by Ang-2 and is in line with a previous report suggesting that in contrast to Ang-2, Ang-1 increases the glycocalyx depth and reduces vascular permeability [31, 49]. To further elaborate the permeability in GOF mice, we analyzed the permeability of circulating HRP tracer by EM. We observed a greater number of HRP-positive vesicles in Ang-2 GOF mice compared to WT mice (Fig. 5b; Suppl. Table 3). These data indicate that Ang-2 could lead to transcellular vesicular permeability and are in line with previous work showing increased caveolae-mediated transport upon pericyte loss and subsequent permeability in genetic mouse models [4]. Western blots of isolated mouse brain microvessels (MBMV) revealed a downregulation of VE-cadherin and claudin-5 in GOF animals, whereas caveolin-1, crucial for caveolae formation was upregulated (Fig. 5c). These data support the EM data that indicated increased numbers of vesicles and gaps in junctions (Figs. 4, 5b; Suppl. Fig. 2a). Glut-1 and β-catenin were however unaltered (Fig. 5c). However, mRNA levels of MECA 32, a plasmalemma vesicle-associated protein (PLVAP), and Mfsd2a that is associated with increased vesicular formation upon pericyte degeneration, were not altered (Suppl. Fig. 4a). Further, flow cytometry analyses revealed an increased number of myeloid cells in brains derived from Ang-2 GOF mice compared to WT mice (Suppl. Fig. 3a), a finding that is consistent with previous observations [52]. Together, these data indicate an effect of Ang-2 on both paracellular and transcellular permeability.Fig. 3


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)

Detection of the glycocalyx and plasma tracers by EM analysis: a lanthanum nitrate was used to detect the glycocalyx (black arrows) in mouse brain vessels. Glycocalyx was decreased/disrupted from ~300 to ~100 nm in GOF mice. b HRP was intravenously injected into WT and Ang-2 GOF mice and circulated for 30 min. Representative images of Ang-2 GOF mice revealed HRP-vesicles (black arrows) with affected endothelial junctions (red arrows). Quantitative analysis indicated more HRP-positive vesicles in Ang-2 GOF mice (20 vessels analyzed; n = 3; scale bar sizes are indicated in the images). c MBMV Western blots showed decreased VE-cadherin and claudin-5 in GOF, whereas caveolin-1 was upregulated (n = 3)
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Fig5: Detection of the glycocalyx and plasma tracers by EM analysis: a lanthanum nitrate was used to detect the glycocalyx (black arrows) in mouse brain vessels. Glycocalyx was decreased/disrupted from ~300 to ~100 nm in GOF mice. b HRP was intravenously injected into WT and Ang-2 GOF mice and circulated for 30 min. Representative images of Ang-2 GOF mice revealed HRP-vesicles (black arrows) with affected endothelial junctions (red arrows). Quantitative analysis indicated more HRP-positive vesicles in Ang-2 GOF mice (20 vessels analyzed; n = 3; scale bar sizes are indicated in the images). c MBMV Western blots showed decreased VE-cadherin and claudin-5 in GOF, whereas caveolin-1 was upregulated (n = 3)
Mentions: We next elaborated the molecular components of Ang-2-mediated permeability by immunohistochemistry (IHC), electron microscopy (EM) and Western blots. Quantitative RT-PCR analysis confirmed an overexpression of hAng-2 in the isolated brain microvessels of Ang-2 GOF mice, whereas Tie2 expression levels were not altered (Suppl. Fig. 1d). Furthermore, we did not observe an increase in Ang-1 levels (Suppl. Fig. 1e, f) in these mice, suggesting an absence of its compensatory effects. IHC from cryosections (10 µm) and from vibratome sections (50 µm) revealed a decrease in pericytes in GOF (Fig. 3a, b; Suppl. video) as previously observed in the periphery [53]. In Fig. 3a, we set the WT pericyte coverage to 100 %; however, the absolute pericyte coverage of the vessels is only about 20–25 % as obtained from vibratome data analysis which is reduced to approximately 12–15 % in the GOF mice. Also by EM analysis, we observed decreased pericyte coverage with increased numbers of degenerating pericytes in Ang-2 GOF mice (Fig. 4a–c; Suppl. Table 1). EM analysis also showed increased caveolae-like vesicles (Fig. 4a, e) that are partly responsible for peripheral endothelial permeability. The analysis also revealed decreased complexity of the inter-endothelial junctions in GOF, with frequent gaps (Fig. 4a, d; Suppl. Fig. 2a; Suppl. Table 2), indicating increased paracellular permeability. Furthermore, the glycocalyx thickness was considerably disrupted/decreased (~300–~100 nm, Fig. 5a; Suppl. Fig. 2b). This finding implies increased permeability induced by Ang-2 and is in line with a previous report suggesting that in contrast to Ang-2, Ang-1 increases the glycocalyx depth and reduces vascular permeability [31, 49]. To further elaborate the permeability in GOF mice, we analyzed the permeability of circulating HRP tracer by EM. We observed a greater number of HRP-positive vesicles in Ang-2 GOF mice compared to WT mice (Fig. 5b; Suppl. Table 3). These data indicate that Ang-2 could lead to transcellular vesicular permeability and are in line with previous work showing increased caveolae-mediated transport upon pericyte loss and subsequent permeability in genetic mouse models [4]. Western blots of isolated mouse brain microvessels (MBMV) revealed a downregulation of VE-cadherin and claudin-5 in GOF animals, whereas caveolin-1, crucial for caveolae formation was upregulated (Fig. 5c). These data support the EM data that indicated increased numbers of vesicles and gaps in junctions (Figs. 4, 5b; Suppl. Fig. 2a). Glut-1 and β-catenin were however unaltered (Fig. 5c). However, mRNA levels of MECA 32, a plasmalemma vesicle-associated protein (PLVAP), and Mfsd2a that is associated with increased vesicular formation upon pericyte degeneration, were not altered (Suppl. Fig. 4a). Further, flow cytometry analyses revealed an increased number of myeloid cells in brains derived from Ang-2 GOF mice compared to WT mice (Suppl. Fig. 3a), a finding that is consistent with previous observations [52]. Together, these data indicate an effect of Ang-2 on both paracellular and transcellular permeability.Fig. 3

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