Single-cell atlas of the human brain vasculature across development, adulthood and disease.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature.

Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.

Republication: Targeting PI3KC2ß Impairs Proliferation and Survival in Acute Leukemia, Brain Tumours and Neuroendocrine Tumours.

BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2ß isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2ß was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2ß or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2ß also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2ß contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.

Tunneling nanotubes evoke pericyte/endothelial communication during normal and tumoral angiogenesis.

BACKGROUND: Nanotubular structures, denoted tunneling nanotubes (TNTs) have been described in recent times as involved in cell-to-cell communication between distant cells. Nevertheless, TNT-like, long filopodial processes had already been described in the last century as connecting facing, growing microvessels during the process of cerebral cortex vascularization and collateralization. Here we have investigated the possible presence and the cellular origin of TNTs during normal brain vascularization and also in highly vascularized brain tumors. METHODS: We searched for TNTs by high-resolution immunofluorescence confocal microscopy, applied to the analysis of 20-µm, thick sections from lightly fixed, unembedded samples of both developing cerebral cortex and human glioblastoma (GB), immunolabeled for endothelial, pericyte, and astrocyte markers, and vessel basal lamina molecules. RESULTS: The results revealed the existence of pericyte-derived TNTs, labeled by proteoglycan NG2/CSPG4 and CD146. In agreement with the described heterogeneity of these nanostructures, ultra-long (> 300 µm) and very thin (< 0.8 µm) TNTs were observed to bridge the gap between the wall of distant vessels, or were detected as short (< 300 µm) bridging cables connecting a vessel sprout with its facing vessel or two apposed vessel sprouts. The pericyte origin of TNTs ex vivo in fetal cortex and GB was confirmed by in vitro analysis of brain pericytes, which were able to form and remained connected by typical TNT structures. CONCLUSIONS: None of the multiple roles described for TNTs can be excluded from a possible involvement during the processes of both normal and pathological vessel growth. A possible function, suggested by the pioneering studies made during cerebral cortex vascularization, is in cell searching and cell-to-cell recognition during the processes of vessel collateralization and vascular network formation. According to our results, it is definitely the pericyte-derived TNTs that seem to actively explore the surrounding microenvironment, searching for (site-to-site recognition), and connecting with (pericyte-to-pericyte and/or pericyte-to-endothelial cell communication), the targeted vessels. This idea implies that TNTs may have a primary role in the very early phases of both physiological and tumor angiogenesis in the brain.

Age-associated and therapy-induced alterations in the cellular microenvironment of experimental gliomas.

The poor prognosis associated with advanced age in patients with glioblastoma remains poorly understood. Glioblastoma in the elderly has been particularly associated with vascular endothelial growth factor (VEGF)-dependent angiogenesis, and early uncontrolled studies suggested that the anti-angiogenic agent bevacizumab (BEV), an antibody to VEGF, might be preferentially active in this patient population. Accordingly, we explored host age-dependent differences in survival and benefit from radiotherapy (RT) or BEV in syngeneic mouse glioma models. Survival was inferior in older mice in the SMA-540 and and less so in SMA-560, but not in the SMA-497 or GL-261 models. Detailed flow cytometric studies revealed increased myeloid and decreased effector T cell population frequencies in SMA-540 tumors of old compared to young mice, but no such difference in the SMA-497 model. Bone marrow transplantation (BMT) from young to old mice had no effect, whereas survival was reduced with BMT from old to young mice. BEV significantly decreased vessel densities in gliomas of old, but not young mice. Accordingly, old, but not young SMA-540 tumor-bearing mice benefited from BEV alone or in combination with RT. End-stage tumors of old BEV- and BEV/RT-treated mice exhibited increased infiltration of T helper and cytotoxic T cells compared to tumors of young mice. The SMA-540 model may provide a valuable tool to evaluate the influence of host age on glioblastoma progression and treatment response. The biological host factors that modulate glioma growth in old as opposed to young mice remain to be identified.

Interleukin-33 in human gliomas: Expression and prognostic significance.

Interleukin-33 (IL-33) is a nuclear and pleiotropic cytokine with regard to its cellular sources and its actions. IL-33 is involved in the pathogenesis of brain diseases. Several factors account for the tumorigenicity of human gliomas, including cytokines and their receptors. The present study assessed the expression and prognostic significance of IL-33 in human astroglial brain tumors. Protein levels of IL-33 were determined by immunohistochemistry using a tissue microarray containing 95 human gliomas. mRNA expression data of IL-33, as well as of its receptors, IL-1 receptor-like 1 protein and IL-1 receptor accessory protein (IL1RAcP), were obtained from The Cancer Genome Atlas database. IL-33 protein was expressed heterogeneously in tumor tissue, but was, however, not detected in normal brain tissue. There was no differential IL-33 protein expression by tumor grade, while IL-33 protein expression was associated with inferior survival in patients with recurrent glioblastomas. Interrogations of the TCGA database indicated that mRNA expression of IL-33 and the IL-33 receptors was heterogeneous, and that IL-33 and IL1RAcP mRNA levels were correlated with the tumor grade. Elevated IL-33 mRNA levels were associated with the inferior survival of glioblastoma patients. Therefore, IL-33 may play an important role in the pathogenesis and prognosis of human gliomas.

Wiring the Vascular Network with Neural Cues: A CNS Perspective.

The vascular and the nervous system are responsible for oxygen, nutrient, and information transfer and thereby constitute highly important communication systems in higher organisms. These functional similarities are reflected at the anatomical, cellular, and molecular levels, where common developmental principles and mutual crosstalks have evolved to coordinate their action. This resemblance of the two systems at different levels of complexity has been termed the "neurovascular link." Most of the evidence demonstrating neurovascular interactions derives from studies outside the CNS and from the CNS tissue of the retina. However, little is known about the specific properties of the neurovascular link in the brain. Here, we focus on regulatory effects of molecules involved in the neurovascular link on angiogenesis in the periphery and in the brain and distinguish between general and CNS-specific cues for angiogenesis. Moreover, we discuss the emerging molecular interactions of these angiogenic cues with the VEGF-VEGFR-Delta-like ligand 4 (Dll4)-Jagged-Notch pathway.

Modulation of cerebral endothelial cell function by TGF-ß in glioblastoma: VEGF-dependent angiogenesis versus endothelial mesenchymal transition.

Glioblastoma are among the most angiogenic tumors. The molecular mechanisms that control blood vessel formation by endothelial cells (EC) in glioblastoma remain incompletely understood. Transforming growth factor-ß (TGF-ß) is a key regulatory cytokine that has proinvasive and stemness-maintaining autocrine properties in glioblastoma and confers immunosuppression to the tumor microenvironment. Here we characterize potential pro- and anti-angiogenic activities of TGF-ß in the context of glioblastoma in vitro, using human brain-derived microvascular endothelial cells (hCMEC/D3) and glioblastoma-derived endothelial cells (GMEC) as model systems. We find that TGF-ß induces vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) mRNA expression and protein release in a TGF-ß receptor (TßR) II / activin-like kinase (ALK)-5-dependent manner under normoxia and hypoxia, defining potential indirect proangiogenic activity of TGF-ß in glioblastoma. In parallel, exogenous TGF-ß has also inhibitory effects on EC properties and induces endothelial-mesenchymal transition (EndMT) in hCMEC and GMEC. Accordingly, direct inhibition of endogenous TGF-ß/ALK-5 signalling increases EC properties such as tube formation, von-Willebrand factor (vWF) and claudin (CLDN) 5 expression. Yet, the supernatant of TGF-ß-stimulated hCMEC and GMEC strongly promotes EC-related gene expression and tube formation in a cediranib-sensitive manner. These observations shed light on the complex pro- and anti-angiogenic pathways involving the cross-talk between TGF-ß and VEGF/PLGF signalling in glioblastoma which may involve parallel stimulation of angiogenesis and EndMT in distinct target cell populations.

Transforming growth factor-ß pathway activity in glioblastoma.

Transforming growth factor (TGF)-ß is a central molecule maintaining the malignant phenotype of glioblastoma. Anti-TGF-ß strategies are currently being explored in early clinical trials. Yet, there is little contemporary data on the differential expression of TGF-ß isoforms at the mRNA and protein level or TGF-ß/Smad pathway activity in glioblastomas in vivo.Here we studied 64 newly diagnosed and 16 recurrent glioblastomas for the expression of TGF-ß1-3, platelet-derived growth factor (PDGF)-B, and plasminogen activator inhibitor (PAI)-1 mRNA by RT-PCR and for the levels of TGF-ß1-3 protein, phosphorylated Smad2 (pSmad2), pSmad1/5/8 and PAI-1 by immunohistochemistry.Among the TGF-ß isoforms, TGF-ß1 mRNA was the most, whereas TGF-ß3 mRNA was the least abundant. TGF-ß1-3 mRNA expression was strongly correlated, as was the expression of TGF-ß1-3 mRNA, and of the TGF-ß1-3 target genes, PDGF-B and PAI-1. TGF-ß2 and TGF-ß3 protein levels correlated well, whereas the comparison of the other TGF-ßisoforms did not. Positive correlation was also observed between TGF-ß1 and pSmad1/5/8 and between pSmad2 and pSmad1/5/8. Survival analyses indicated that a group of patients with high expression levels of TGF-ß2 mRNA or pSmad1/5/8 protein have inferior outcome.We thus provide potential biomarkers for patient stratification in clinical trials of anti-TGF-ß therapies in glioblastoma.

How stemlike are sphere cultures from long-term cancer cell lines? Lessons from mouse glioma models.

Cancer stem cells may mediate therapy resistance and recurrence in various types of cancer, including glioblastoma. Cancer stemlike cells can be isolated from long-term cancer cell lines, including glioma lines. Using sphere formation as a model for cancer cell stemness in vitro, we derived sphere cultures from SMA-497, SMA-540, SMA-560, and GL-261 glioma cells. Gene expression and proteomics profiling demonstrated that sphere cultures uniformly showed an elevated expression of stemness-associated genes, notably including CD44. Differences in neural lineage marker expression between nonsphere and sphere cultures were heterogeneous except for a uniform reduction of ß-III-tubulin in sphere cultures. All sphere cultures showed slower growth. Self-renewal capacity was influenced by medium conditions but not nonsphere versus sphere culture phenotype. Sphere cultures were more resistant to irradiation, whereas both nonsphere and sphere cultures were highly resistant to temozolomide. Nonsphere cells formed more aggressive tumors in syngeneic mice than sphere cells in all models except SMA-560. There were no major differences in vascularization or infiltration by T cells or microglia/macrophages between nonsphere and sphere cell-derived tumors implanted in syngeneic hosts. Together, these data indicate that mouse glioma cell lines may be induced in vitro to form spheres that acquire features of stemness, but they do not exhibit a uniform biologic phenotype, thereby challenging the view that they represent a superior model system.

Redox-directed cancer therapeutics: Taurolidine and Piperlongumine as broadly effective antineoplastic agents (review).

Targeting the oxygen stress response pathway is considered a promising strategy to exert antineoplastic activity in a broad spectrum of tumor types. Supporting this view, we summarize the mechanism of action of Taurolidine and Piperlongumine, two antineoplastic agents with strikingly broad tumor selectivity. Taurolidine enhances the oxidative stress (ROS) selectively in tumor cells. Its cytotoxicity for various tumor cells in vitro and in vivo, which includes tumor stem cells, is based on the induction of programmed cell death, largely via apoptosis but also necroptosis and autophagy. The redox-directed mechanism of action of Taurolidine is apparent from the finding that reducing agents e.g., N-acetylcysteine or glutathione impair its cytotoxicity, while its effectiveness is enhanced by agents which inhibit the cellular anti­oxidant capacity. A similar redox-directed antineoplastic action is shown by Piperlongumine, a recently described experimental drug of plant origin. Taurolidine is particularly advantageous in surgical oncology as this taurine-derivative can be applied perioperatively or systemically with good tolerability as shown in initial clinical applications.

Bleeding propensity of cavernous malformations: impact of tight junction alterations on the occurrence of overt hematoma.

OBJECT: Endothelial tight junction (TJ) expression is mostly absent in cerebral cavernous malformations (CMs), which causes increased perilesional erythrocyte and fluid oozing. However, in a subset of CM lesions, foci of preserved TJ staining are observed along endothelial cell contacts. The clinical relevance of this finding is unclear. This study investigates the relevance of the focal TJ protein expression and its association with CM bleeding propensity. METHODS: Immunohistochemical staining for the TJ proteins claudin-5, occludin, and ZO-1 was performed on 32 CM specimens that were resected during 2008-2010. The patients were allocated to 2 groups according to TJ protein expression, and the clinical and radiological parameters of aggressiveness were analyzed and compared. RESULTS Complete absence of TJ expression was identified in 20 specimens, and focal TJ protein expression in 12. CMs without TJ immunoreactivity were significantly larger (p = 0.022) and had a significantly greater propensity for development of frank hematomas (p = 0.028) and perilesional edema (p = 0.013). Symptom severity, multiplicity, developmental venous anomaly (DVA) presence, and CM location did not show a significant difference depending on TJ expression. CONCLUSIONS: In a univariate analysis the authors observed significantly less propensity for frank hematomas and perilesional edema as well as smaller size in CM lesions with focal TJ expression compared with CMs without TJ expression. The observed difference in TJ protein expression might be the reason for differences in bleeding propensity of the CM lesions. Although this finding cannot be used in predictive manner at this time, it is a basis for further multivariate analyses of possible CM biological predictors.

Targeting class IA PI3K isoforms selectively impairs cell growth, survival, and migration in glioblastoma.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110α was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110ß expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110α/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110α activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110α or PI3K p110ß also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110δ did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110α can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110α/p-S6.

Interferon-ß induces loss of spherogenicity and overcomes therapy resistance of glioblastoma stem cells.

Glioblastoma is the most common malignant brain tumor in adults and characterized by a poor prognosis. Glioma cells expressing O(6)-methylguanine DNA methyltransferase (MGMT) exhibit a higher level of resistance toward alkylating agents, including the standard of care chemotherapeutic agent temozolomide. Here, we demonstrate that long-term glioma cell lines (LTL) as well as glioma-initiating cell lines (GIC) express receptors for the immune modulatory cytokine IFN-ß and respond to IFN-ß with induction of STAT-3 phosphorylation. Exposure to IFN-ß induces a minor loss of viability, but strongly interferes with sphere formation in GIC cultures. Furthermore, IFN-ß sensitizes LTL and GIC to temozolomide and irradiation. RNA interference confirmed that both IFN-ß receptors, R1 and R2, are required for IFN-ß-mediated sensitization, but that sensitization is independent of MGMT or TP53. Most GIC lines are highly temozolomide-resistant, mediated by MGMT expression, but nevertheless susceptible to IFN-ß sensitization. Gene expression profiling following IFN-ß treatment revealed strong upregulation of IFN-ß-associated genes, including a proapoptotic gene cluster, but did not alter stemness-associated expression signatures. Caspase activity and inhibition studies revealed the proapoptotic genes to mediate glioma cell sensitization to exogenous death ligands by IFN-ß, but not to temozolomide or irradiation, indicating distinct pathways of death sensitization mediated by IFN-ß. Thus, IFN-ß is a potential adjunct to glioblastoma treatment that may target the GIC population. IFN-ß operates independently of MGMT-mediated resistance, classical apoptosis-regulatory networks, and stemness-associated gene clusters.

Thymosin ß 4 gene silencing decreases stemness and invasiveness in glioblastoma.

Thymosin beta 4 is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. Thymosin beta 4 gene silencing promotes differentiation of neural stem cells whereas thymosin beta 4 overexpression initiates cortical folding of developing brain hemispheres. A role of thymosin beta 4 in malignant gliomas has not yet been investigated. We analysed thymosin beta 4 staining on tissue microarrays and performed interrogations of the REMBRANDT and the Cancer Genome Atlas databases. We investigated thymosin beta 4 expression in seven established glioma cell lines and seven glioma-initiating cell lines and induced or silenced thymosin beta 4 expression by lentiviral transduction in LNT-229, U87MG and GS-2 cells to study the effects of altered thymosin beta 4 expression on gene expression, growth, clonogenicity, migration, invasion, self-renewal and differentiation capacity in vitro, and tumorigenicity in vivo. Thymosin beta 4 expression increased with grade of malignancy in gliomas. Thymosin beta 4 gene silencing in LNT-229 and U87MG glioma cells inhibited migration and invasion, promoted starvation-induced cell death in vitro and enhanced survival of glioma-bearing mice. Thymosin beta 4 gene silencing in GS-2 cells inhibited self-renewal and promoted differentiation in vitro and decreased tumorigenicity in vivo. Gene expression analysis suggested a thymosin beta 4-dependent regulation of mesenchymal signature genes and modulation of TGFß and p53 signalling networks. We conclude that thymosin beta 4 should be explored as a novel molecular target for anti-glioma therapy.

Nogo-A is a negative regulator of CNS angiogenesis.

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A-specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti-Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

Soluble α-klotho: a novel serum biomarker for the activity of GH-producing pituitary adenomas.

OBJECTIVE: Klotho is a lifespan-influencing gene expressed mainly in the kidneys. Soluble α-Klotho (αKL) is released into the circulation. In this study, we present baseline αKL serum levels of patients with acromegaly compared with controls with other pituitary adenomas and assess changes following transsphenoidal surgery. DESIGN: Prospective controlled study. METHODS: We measured soluble αKL (sandwich ELISA) and IGF1 (RIA) in sera of 14 patients (eight females and six males) with active acromegaly and in 22 control patients (13 females and nine males) operated for non-GH-producing pituitary adenomas. Immunohistochemical staining for Klotho was performed in resected adenomas and in normal pituitary tissue samples. RESULTS: Soluble αKL was high in the acromegaly group preoperatively (median 4217 pg/ml, interquartile range (IQR) 1812-6623 pg/ml) and declined after surgery during early follow-up (2-6 days; median 645 pg/ml, IQR 550-1303 pg/ml) (P<0.001) and during late follow-up (2-3 months post-operatively; median 902 pg/ml, IQR 497-1340 pg/ml; P<0.001). In controls, preoperative soluble αKL was significantly lower than in acromegalics, 532 pg/ml (400-677 pg/ml; P<0.001). Following surgery, soluble αKL remained low during early and late follow-up - changes over time within the control group were not statistically significant. These results were independent of age, sex and kidney function. Klotho staining was equal or slightly decreased in GH-positive adenomas compared with controls. CONCLUSION: High soluble αKL serum levels were specific to GH-producing adenomas and decreased rapidly following adenoma removal. Thus, soluble αKL appears to be a new specific and sensitive biomarker reflecting disease activity in acromegaly. Similar Klotho staining patterns in controls and acromegalics suggest that the rise in serum αKL is caused by systemic actions of pituitary GH rather than due to increased expression of Klotho by the pituitary (adenoma).

The CXCL12/CXCR4/CXCR7 ligand-receptor system regulates neuro-glio-vascular interactions and vessel growth during human brain development.

This study investigates glio-vascular interactions in human fetal brain at midgestation, specifically examining the expression and immunolocalization of the CXCL12/CXCR4/CXCR7 ligand-receptor axis and its possible role in the vascular patterning of the developing brain. At midgestation, the telencephalic vesicles are characterized by well developed radial glia cells (RGCs), the first differentiated astrocytes and a basic vascular network mainly built of radial vessels. RGCs have been recognized to contribute to cerebral cortex neuro-vascular architecture and have also been demonstrated to act as a significant source of neural cells (Rakic, Brain Res 33:471-476, 1971; Malatesta et al, Development 127:5253-5263, 2000). According to our hypothesis CXCL12, a potent migration and differentiation chemokine released by RGCs, may act as a linking factor coordinating neuroblast migration with vessel growth and patterning through the activation of different ligand/receptor axes. The obtained results support this hypothesis showing that together with CXCR4/CXCR7-reactive neuroblasts, which migrate in close association with CXCL12 RGCs, layer-specific subsets of CXCL12 RGCs and astrocytes specifically contact the microvessel wall. Moreover, the CXCL12/CXCR4/CXCR7 system appears to be directly involved in microvessel growth, its members being differentially expressed in angiogenically activated microvessels and vascular sprouts.

Diversified expression of NG2/CSPG4 isoforms in glioblastoma and human foetal brain identifies pericyte subsets.

NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating between phenotype-specific and neoplastic versus non-neoplastic variants of the PG, thus opening up vistas for more selective immunotherapeutic targeting of brain tumours.

Stroma-derived interleukin-34 controls the development and maintenance of langerhans cells and the maintenance of microglia.

Colony stimulating factor-1 (Csf-1) receptor and its ligand Csf-1 control macrophage development, maintenance, and function. The development of both Langerhans cells (LCs) and microglia is highly dependent on Csf-1 receptor signaling but independent of Csf-1. Here we show that in both mice and humans, interleukin-34 (IL-34), an alternative ligand for Csf-1 receptor, is produced by keratinocytes in the epidermis and by neurons in the brain. Mice lacking IL-34 displayed a marked reduction of LCs and a decrease of microglia, whereas monocytes, dermal, and lymphoid tissue macrophages and DCs were unaffected. We identified IL-34 as a nonredundant cytokine for the development of LCs during embryogenesis as well as for their homeostasis in the adult skin. Whereas inflammation-induced repopulation of LCs appears to be dependent on Csf-1, once inflammation is resolved, LC survival is again IL-34-dependent. In contrast, microglia and their yolk sac precursors develop independently of IL-34 but rely on it for their maintenance in the adult brain.