Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood-brain barrier in acute ischemic stroke.

Blood-brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database ( https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke ). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

New Fully Automated Preparation of High Apparent Molar Activity 68Ga-FAPI-46 on a Trasis AiO Platform.

A large number of applications for fibroblast activation protein inhibitors (FAPI)-based PET agents have been evaluated in conditions ranging from cancer to non-malignant diseases such as myocardial infarction. In particular, 68Ga-FAPI-46 was reported to have a high specificity and affinity for FAP-expressing cells, a fast and high accumulation in tumor lesions/injuries together with a fast body clearance when investigated in vivo. Due to the increasing interest in the use of the agent both preclinically and clinically, we developed an automated synthesis for the production of 68Ga-FAPI-46 on a Trasis AiO platform. The new synthetic procedure, which included the processing of the generator eluate using a strong cation exchange resin and a final purification step through an HLB followed by a QMA cartridge, yielded 68Ga-FAPI-46 with high radiochemical purity (>98%) and apparent molar activity (271.1 ± 105.6 MBq/nmol). Additionally, the in vitro and in vivo properties of the product were assessed on glioblastoma cells and mouse model. Although developed for the preparation of 68Ga-FAPI-46 for preclinical use, our method can be adapted for clinical production as a reliable alternative to the manual (i.e., cold kit) or modular systems preparations already described in the literature.

Role of angiopoietin-2 in venous thrombus resolution and chronic thromboembolic disease.

BACKGROUND: Defective angiogenesis, incomplete thrombus revascularisation and fibrosis are considered critical pathomechanisms of chronic thromboembolic pulmonary hypertension (CTEPH) after pulmonary embolism. Angiopoietin-2 (ANGPT2) has been shown to regulate angiogenesis, but its importance for thrombus resolution and remodelling is unknown. METHODS: ANGPT2 plasma concentrations were measured in patients with CTEPH (n=68) and acute pulmonary embolism (n=84). Tissue removed during pulmonary endarterectomy (PEA) for CTEPH was analysed (immuno)histologically. A mouse model of inferior vena cava ligation was used to study the kinetics of venous thrombus resolution in wild-type mice receiving recombinant ANGPT2 via osmotic pumps, and in transgenic mice overexpressing ANGPT2 in endothelial cells. RESULTS: Circulating ANGPT2 levels were higher in CTEPH patients compared to patients with idiopathic pulmonary arterial hypertension and healthy controls, and decreased after PEA. Plasma ANGPT2 levels were elevated in patients with pulmonary embolism and diagnosis of CTEPH during follow-up. Histological analysis of PEA specimens confirmed increased ANGPT2 expression, and low levels of phosphorylated TIE2 were observed in regions with early-organised pulmonary thrombi, myofibroblasts and fibrosis. Microarray and high-resolution microscopy analysis could localise ANGPT2 overexpression to endothelial cells, and hypoxia and transforming growth factor-ß1 were identified as potential stimuli. Gain-of-function experiments in mice demonstrated that exogenous ANGPT2 administration and transgenic endothelial ANGPT2 overexpression resulted in delayed venous thrombus resolution, and thrombi were characterised by lower TIE2 phosphorylation and fewer microvessels. CONCLUSION: Our findings suggest that ANGPT2 delays venous thrombus resolution and that overexpression of ANGPT2 contributes to thrombofibrosis and may thus support the transition from pulmonary embolism to CTEPH.

Dexamethasone-induced leukocytosis is associated with poor survival in newly diagnosed glioblastoma.

Despite its well-characterized side effects, dexamethasone is widely used in the pre-, peri- and postoperative neurosurgical setting due to its effective relief of tumor-induced symptoms through the reduction of tumor-associated edema. However, some patients show laboratory-defined dexamethasone induced elevation of white blood cell count, and its impact on glioblastoma progression is unknown. We retrospectively analyzed 113 patients with newly diagnosed glioblastoma to describe the incidence, risk factors and clinical features of dexamethasone-induced leukocytosis in primary glioblastoma patients. We further conducted an immunohistochemical analysis of the granulocyte and lymphocyte tumor-infiltration in the available corresponding histological sections. Patient age was identified to be a risk factor for the development of dexamethasone-induced leukocytosis (p < 0.05). The presence of dexamethasone-induced leukocytosis decreased overall survival (HR 2.25 95% CI [1.15-4.38]; p < 0.001) and progression-free survival (HR 2.23 95% CI [1.09-4.59]; p < 0.01). Furthermore, patients with dexamethasone-induced leukocytosis had significantly reduced CD15 + granulocytic- (p < 0.05) and CD3 + lymphocytic tumour infiltration (p < 0.05). We identified a subgroup of glioblastoma patients that are at particularly high risk for poor outcome upon dexamethasone treatment. Therefore, restrictive dosage or other edema reducing substances should be considered in patients with dexamethasone-induced leukocytosis.

Paired box gene 8 (PAX8) expression is associated with sonic hedgehog (SHH)/wingless int (WNT) subtypes, desmoplastic histology and patient survival in human medulloblastomas.

AIMS: The paired box gene 8 (PAX8) plays crucial roles in organ patterning and cellular differentiation during development and tumorigenesis. Although its function is partly understood in vertebrate development, there is poor data concerning human central nervous system (CNS) development and brain tumours. METHODS: We investigated developing human (n = 19) and mouse (n = 3) brains as well as medulloblastomas (MBs) (n = 113) for PAX8 expression by immunohistochemistry. Human MB cell lines were assessed for PAX8 expression using polymerase chain reaction and immunoblotting and analysed for growth and migration following PAX8 knock-down by small interfering ribonucleic acid (siRNA). RESULTS: PAX8 protein expression was associated with germinal layers in human and murine forebrain and hindbrain development. PAX8 expression significantly decreased over time in the external granule cell layer but increased in the internal granule cell layer. In MB subtypes, we observed an association of PAX8 expression with sonic hedgehog (SHH) and wingless int subtypes but not with group 3 and 4 MBs. Beyond that, we detected high PAX8 levels in desmoplastic MB subtypes. Univariate analyses revealed high PAX8 levels as a prognostic factor associated with a significantly better patient prognosis in human MB (overall survival: Log-Rank P = 0.0404, Wilcoxon P = 0.0280; progression-free survival: Log-Rank P = 0.0225; Wilcoxon P = 0.0136). In vitro assays revealed increased proliferation and migration of MB cell lines after PAX8 siRNA knock-down. CONCLUSION: In summary, high PAX8 expression is linked to better prognosis in MBs potentially by suppressing both proliferative and migratory properties of MB cells. The distinct spatio-temporal expression pattern of PAX8 during brain development might contribute to the understanding of distinct MB subtype histogenesis.

Anti-osteopontin therapy leads to improved edema and infarct size in a murine model of ischemic stroke.

Ischemic stroke is a serious neurological disorder that is associated with dysregulation of the neurovascular unit (NVU) and impairment of the blood-brain barrier (BBB). Paradoxically, reperfusion therapies can aggravate NVU and BBB dysfunction, leading to deleterious consequences in addition to the obvious benefits. Using the recently established EPAM-ia method, we identified osteopontin as a target dysregulated in multiple NVU cell types and demonstrated that osteopontin targeting in the early acute phase post-transient middle cerebral artery occlusion (tMCAO) evolves protective effects. Here, we assessed the time course of osteopontin and CD44 receptor expression in NVU cells and examined cerebroprotective effects of osteopontin targeting in early and late acute phases of ischemic stroke. Expression analysis of osteopontin and CD44 receptor post-tMCAO indicated increased levels of both, from early to late acute phases, which was supported by their co-localization in NVU cells. Combined osteopontin targeting in early and late acute phases with anti-osteopontin antibody resulted in further improvement in BBB recovery and edema reduction compared to targeting only in the early acute phase comprising the reperfusion window. Combined targeting led to reduced infarct volumes, which was not observed for the single early acute phase targeting. The effects of the therapeutic antibody were confirmed both in vitro and in vivo in reducing osteopontin and CD44 expression. Osteopontin targeting at the NVU in early and late acute phases of ischemic stroke improves edema and infarct size in mice, suggesting anti-osteopontin therapy as promising adjunctive treatment to reperfusion therapy.

Low wnt/ß-catenin signaling determines leaky vessels in the subfornical organ and affects water homeostasis in mice.

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/ß-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 ß-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.

Upregulation of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha in leptomeningeal vascular malformations of Sturge-Weber syndrome.

Cutaneous and leptomeningeal vascular malformations are hallmarks of the Sturge-Weber Syndrome (SWS), resulting in chronic ischemic tissue damage. The mechanisms underlying the pathobiology of these progressive lesions are unknown. Aberrant expression of angiogenic factors has been implicated in the genesis and maintenance of vascular malformations. To assess the role of angiogenesis in SWS vascular lesions we determined the expression of key angiogenic factors by immunohistochemistry and in situ hybridization in 8 SWS patients (age: 8 months to 18 years). We observed increased expression of vascular endothelial growth factor (VEGF), its cognate receptors VEGFR-1, VEGFR-2, and neuropilin (NP)-1 as well as Tie2 in leptomeningeal SWS blood vessels. Intriguingly, these factors are known to be transcriptionally induced by hypoxia-inducible factor (HIF). The HIF system has emerged as the key regulatory system of responses to hypoxia. Immunohistochemical analysis demonstrated markedly elevated nuclear HIF-1alpha and HIF-2alpha protein levels in SWS vessels. Concomitantly, SWS vessels revealed signs of enhanced endothelial cell (EC) turnover as evidenced by increased EC proliferation and apoptosis. Thus, in terms of angiogenesis, vascular malformations in SWS are not static lesions but constitute dynamic structures. Our observation of a dysregulated HIF-alpha expression in SWS vessels are in agreement with recent findings that EC-specific HIF activation provides a setting which supports and sustains angiogenesis and could be of potential use for developing therapeutic strategies to treat these currently incurable lesions.

Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas.

Recently, the conserved intracellular digestion mechanism 'autophagy' has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas.

EGFL7 meets miRNA-126: an angiogenesis alliance.

Blood vessels form de novo through the tightly regulated programs of vasculogenesis and angiogenesis. Both processes are distinct but one of the steps they share is the formation of a central lumen, when groups of cells organized as vascular cords undergo complex changes to achieve a tube-like morphology. Recently, a protein termed epidermal growth factor-like domain 7 (EGFL7) was described as a novel endothelial cell-derived factor involved in the regulation of the spatial arrangement of cells during vascular tube assembly. With its impact on tubulogenesis and vessel shape EGFL7 joined the large family of molecules governing blood vessel formation. Only recently, the molecular mechanisms underlying EGFL7's effects have been started to be elucidated and shaping of the extracellular matrix (ECM) as well as Notch signaling might very well play a role in mediating its biological effects. Further, findings in knock-out animal models suggest miR-126, a miRNA located within the egfl7 gene, has a major role in vessel development by promoting VEGF signaling, angiogenesis and vascular integrity. This review summarizes our current knowledge on EGFL7 and miR-126 and we will discuss the implications of both bioactive molecules for the formation of blood vessels.

Wnt/beta-catenin signaling controls development of the blood-brain barrier.

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.

Endothelial cell transplantation for gene therapy in experimental gliomas.

OBJECTIVE: Malignant gliomas are prominent targets for cancer gene therapy approaches because of their poor prognosis despite all available therapies. Endothelial cells (ECs) are considered attractive vehicles for cell-based gene therapy because of their tropism to the tumor vasculature. In this study, we investigated the potential of ECs to incorporate into glioma vessels after intra-arterial or local application to establish whether ECs can be used as cellular vectors for gene therapy in gliomas. METHODS: Immortalized rat brain endothelial cells (BECs) were modified to express either beta-galactosidase or green fluorescent protein (GFP). The ability of transduced BECs to integrate into tumor vessels after interstitial implantation was evaluated in C6 and 9L glioma models. The fate of GFP-BECs was investigated after selective intracarotid injection into C6 tumor-bearing animals. RESULTS: The interstitially grafted BECs organized themselves into vascular-like structures and integrated into the tumor vasculature. Transgene expression was limited to 10 days after injection. After selective intra-arterial injection, numerous GFP-BECs were adherent to the vascular lumen at least 7 days after injection. These cells were evenly distributed within small vessels and capillaries of the injected hemisphere and did not home selectively to the tumor vessels. CONCLUSION: Cell-based therapy approaches to brain tumor treatment using BECs as cellular vectors might be hampered by the rapid downregulation of transgene expression and by the fact that these cells do not home specifically to tumor vessels after intra-arterial injection. Nevertheless, locoregional administration of BECs might be an interesting approach for delivering molecules to brain tumors when short-term expression of transgene in the perivascular space is desirable.