an-vivo-blood-brain-barrier-permeability-assay-mice-using

An <em>In Vivo</em> Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Goethe University Hospital

The formation of new blood vessels plays an important role during the development and progression of a disease. In recent years, there has been a tremendous effort to uncover the molecular mechanisms that drive blood vessel growth in adult tissues. Angiopoietins belong to a family of growth factors that are critically involved in blood vessel formation during developmental and pathological angiogenesis. The importance of Angiopoietin signaling has been recognized in transgenic mouse models as the genetic ablation of Ang-1, and its primary receptor Tie2 has led to early embryonic lethality. Interesting and unusual for a family of ligands, Ang-2 has been identified as an antagonist of Ang-1 in endothelial cells as evidenced by a similar embryonic phenotype when Ang-2 was overexpressed in transgenic mice. In this review, we focus on the functional consequences of autocrine Angiopoietin signaling in endothelial cells.

Angiopoietins.

Glioblastoma multiforme (GBM) is treated by surgical resection followed by radiochemotherapy. Bevacizumab is commonly deployed for anti-angiogenic therapy of recurrent GBM; however, innate immune cells have been identified as instigators of resistance to bevacizumab treatment. We identified angiopoietin-2 (Ang-2) as a potential target in both naive and bevacizumab-treated glioblastoma. Ang-2 expression was absent in normal human brain endothelium, while the highest Ang-2 levels were observed in bevacizumab-treated GBM. In a murine GBM model, VEGF blockade resulted in endothelial upregulation of Ang-2, whereas the combined inhibition of VEGF and Ang-2 leads to extended survival, decreased vascular permeability, depletion of tumor-associated macrophages, improved pericyte coverage, and increased numbers of intratumoral T lymphocytes. CD206(+) (M2-like) macrophages were identified as potential novel targets following anti-angiogenic therapy. Our findings imply a novel role for endothelial cells in therapy resistance and identify endothelial cell/myeloid cell crosstalk mediated by Ang-2 as a potential resistance mechanism. Therefore, combining VEGF blockade with inhibition of Ang-2 may potentially overcome resistance to bevacizumab therapy.

Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma.

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.

Article	Year
Angiopoietins. Recent results in cancer research. Fortschritte der Krebsforschung. Progres dans les recherches sur le cancer\| PubMed ID: 20033375	2010
Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma. EMBO molecular medicine\| PubMed ID: 26666269	2015
Angiopoietin-2-induced blood-brain barrier compromise and increased stroke size are rescued by VE-PTP-dependent restoration of Tie2 signaling. Acta neuropathologica\| PubMed ID: 26932603	2016

Yvonne Reiss

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