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The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/beta-catenin (beta-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of beta-cat in vivo enhances barrier maturation, whereas inactivation of beta-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of beta-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of beta-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of beta-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.
Reproducing the characteristics and the functional responses of the blood–brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.
The tumor vasculature differs from normal blood vessels in morphology, composition and stability. Here, we describe a novel tumor vessel-disrupting mechanism. In an HT1080/mouse xenograft tumor model rhodocetin-αβ was highly effective in disrupting the tumor endothelial barrier. Mechanistically, rhodocetin-αβ triggered MET signaling via neuropilin-1. As both neuropilin-1 and MET were only lumen-exposed in a subset of abnormal tumor vessels, but not in normal vessels, the prime target of rhodocetin-αβ were these abnormal tumor vessels. Consequently, cells lining such tumor vessels became increasingly motile which compromised the vessel wall tightness. After this initial leakage, rhodocetin-αβ could leave the bloodstream and reach the as yet inaccessible neuropilin-1 on the basolateral side of endothelial cells and thus disrupt nearby vessels. Due to the specific neuropilin-1/MET co-distribution on cells lining such abnormal tumor vessels in contrast to normal endothelial cells, rhodocetin-αβ formed the necessary trimeric signaling complex of rhodocetin-αβ-MET-neuropilin-1 only in these abnormal tumor vessels. This selective attack of tumor vessels, sparing endothelial cell-lined vessels of normal tissues, suggests that the neuropilin-1-MET signaling axis may be a promising drugable target for anti-tumor therapy, and that rhodocetin-αβ may serve as a lead structure to develop novel anti-tumor drugs that target such vessels.
Epithelial-to-mesenchymal transition (EMT) is supposed to be responsible for increased invasion and metastases in epithelial cancer cells. The activation of EMT genes has further been proposed to be important in the process of malignant transformation of primary CNS tumors. Since the cellular source and clinical impact of EMT factors in primary CNS tumors still remain unclear, we aimed at deciphering their distribution in vivo and clinico-pathological relevance in human gliomas.
We investigated 350 glioma patients for the expression of the key EMT factors SLUG and TWIST by immunohistochemistry and immunofluorescence related to morpho-genetic alterations such as EGFR-amplification, IDH-1 (R132H) mutation and 1p/19q LOH. Furthermore, transcriptional cluster and survival analyses were performed.
Our data illustrate that SLUG and TWIST are overexpressed in gliomas showing vascular proliferation such as pilocytic astrocytomas and glioblastomas. EMT factors are exclusively expressed by non-neoplastic pericytes/vessel-associated mural cells (VAMCs). They are not associated with patient survival but correlate with pericytic/VAMC genes in glioblastoma cluster analysis.
In summary, the upregulation of EMT genes in pilocytic astrocytomas and glioblastomas reflects the level of activation of pericytes/VAMCs in newly formed blood vessels. Our results underscore that the negative prognostic potential of the EMT signature in the group of diffuse gliomas of WHO grade II-IV does most likely not derive from glioma cells but rather reflects the degree of proliferating mural cells thereby constituting a potential target for future alternative treatment approaches.
Multiple antenatal dexamethasone treatment alters brain vessel differentiation in newborn mouse pups
(2015)
Antenatal steroid treatment decreases morbidity and mortality in premature infants through the maturation of lung tissue, which enables sufficient breathing performance. However, clinical and animal studies have shown that repeated doses of glucocorticoids such as dexamethasone and betamethasone lead to long-term adverse effects on brain development. Therefore, we established a mouse model for antenatal dexamethasone treatment to investigate the effects of dexamethasone on brain vessel differentiation towards the blood-brain barrier (BBB) phenotype, focusing on molecular marker analysis. The major findings were that in total brains on postnatal day (PN) 4 triple antenatal dexamethasone treatment significantly downregulated the tight junction protein claudin-5, the endothelial marker Pecam-1/CD31, the glucocorticoid receptor, the NR1 subunit of the N-methyl-D-aspartate receptor, and Abc transporters (Abcb1a, Abcg2 Abcc4). Less pronounced effects were found after single antenatal dexamethasone treatment and in PN10 samples. Comparisons of total brain samples with isolated brain endothelial cells together with the stainings for Pecam-1/CD31 and claudin-5 led to the assumption that the morphology of brain vessels is affected by antenatal dexamethasone treatment at PN4. On the mRNA level markers for angiogenesis, the sonic hedgehog and the Wnt pathway were downregulated in PN4 samples, suggesting fundamental changes in brain vascularization and/or differentiation. In conclusion, we provided a first comprehensive molecular basis for the adverse effects of multiple antenatal dexamethasone treatment on brain vessel differentiation.
The circumventricular organs (CVOs) in the central nervous system (CNS) lack a vascular blood-brain barrier (BBB), creating communication sites for sensory or secretory neurons, involved in body homeostasis. Wnt/β-catenin signaling is essential for BBB development and maintenance in endothelial cells (ECs) in most CNS vessels. Here we show that in mouse development, as well as in adult mouse and zebrafish, CVO ECs rendered Wnt-reporter negative, suggesting low level pathway activity. Characterization of the subfornical organ (SFO) vasculature revealed heterogenous claudin-5 (Cldn5) and Plvap/Meca32 expression indicative for tight and leaky vessels, respectively. Dominant, EC-specific β-catenin transcription in mice, converted phenotypically leaky into BBB-like vessels, by augmenting Cldn5+ vessels, stabilizing junctions and by reducing Plvap/Meca32+ and fenestrated vessels, resulting in decreased tracer permeability. Endothelial tightening augmented neuronal activity in the SFO of water restricted mice. Hence, regulating the SFO vessel barrier may influence neuronal function in the context of water homeostasis.
The junctional adhesion molecule (JAM)-C is a widely expressed adhesion molecule regulating cell adhesion, cell polarity and inflammation. JAM-C expression and function in the central nervous system (CNS) has been poorly characterized to date. Here we show that JAM-C−/− mice backcrossed onto the C57BL/6 genetic background developed a severe hydrocephalus. An in depth immunohistochemical study revealed specific immunostaining for JAM-C in vascular endothelial cells in the CNS parenchyma, the meninges and in the choroid plexus of healthy C57BL/6 mice. Additional JAM-C immunostaining was detected on ependymal cells lining the ventricles and on choroid plexus epithelial cells. Despite the presence of hemorrhages in the brains of JAM-C−/− mice, our study demonstrates that development of the hydrocephalus was not due to a vascular function of JAM-C as endothelial re-expression of JAM-C failed to rescue the hydrocephalus phenotype of JAM-C−/− C57BL/6 mice. Evaluation of cerebrospinal fluid (CSF) circulation within the ventricular system of JAM-C−/− mice excluded occlusion of the cerebral aqueduct as the cause of hydrocephalus development but showed the acquisition of a block or reduction of CSF drainage from the lateral to the 3rd ventricle in JAM-C−/− C57BL/6 mice. Taken together, our study suggests that JAM-C−/− C57BL/6 mice model the important role for JAM-C in brain development and CSF homeostasis as recently observed in humans with a loss-of-function mutation in JAM-C.
Endothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood–brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin–Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.
Vascularization of the vertebrate brain takes place during embryonic development from a preformed perineural vascular plexus. As a consequence of the intimate contact with neuroectodermal cells the vessels, which are entering the brain exclusively via sprouting angiogenesis, acquire and maintain unique barrier properties known as the blood-brain barrier (BBB). The endothelial BBB depends upon the close association of endothelial cells with pericytes, astrocytes, neurons and microglia, which are summarized in the term neuro-vascular unit. Although it is known since decades that the CNS tissue provides the cues for BBB induction and differentiation in endothelial cells, the molecular mechanism remained obscure. Only recently, the canonical Wnt/beta-catenin pathway and the Wnt7a/7b growth factors have been implicated in brain angiogenesis on the one hand and in BBB induction on the other. This breakthrough in understanding the differentiation of the brain vasculature prompted us to review these findings embedded in the emerging concepts of Wnt signaling in the vasculature. In particular, interactions with other pathways that are crucial for vascular development such as VEGF, Notch, angiopoietins and Sonic hedgehog are discussed. Finally, we considered the potential role of the Wnt pathway in vascular brain pathologies in which BBB function is hampered, as for example in glioma, stroke and Alzheimer's disease.
