Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma.

Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival, these regimens are highly toxic and are associated with significant morbidity. Here, we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas, independent of their subtype. Moreover, high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo, resulting in medulloblastoma regression, decreased metastasis, and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1-and not vascular endothelial growth factor receptor 1-to promote tumor cell survival. This critical tumor-stroma interaction-mediated by Shh, PlGF, and Nrp1 across medulloblastoma subtypes-supports the development of therapies targeting PlGF/Nrp1 pathway.

Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization.

A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.

Surface Tensions of Alkyl Lactates with n-Alkanols or Branched Alkanols.

The surface behavior of 18 binary mixtures at T = 298.15 K and at atmospheric pressure (p = 0.1 MPa) was measured with a drop volume tensiometer. These mixtures are formed by alkyl lactates (methyl lactate and ethyl lactate) and alcohols (n-alkanols from methanol to 1-hexanol and isomeric butanols). Surface tension deviations for all these systems were calculated and correlated with the mole fraction using the Myers-Scott equation. For the mixtures alkyl lactate with methanol, the surface tension deviations are positive. With regard to the mixtures containing ethanol, the surface tension deviation is positive for methyl lactate and slightly negative in the lactate-poor region for ethyl lactate. For the rest of the mixtures, the surface tension deviations are negative. A molecular interpretation of the different behaviors observed was proposed.

Contribution of cell death signaling to blood vessel formation.

The formation of new blood vessels is driven by proliferation of endothelial cells (ECs), elongation of maturing vessel sprouts and ultimately vessel remodeling to create a hierarchically structured vascular system. Vessel regression is an essential process to remove redundant vessel branches in order to adapt the final vessel density to the demands of the surrounding tissue. How exactly vessel regression occurs and whether and to which extent cell death contributes to this process has been in the focus of several studies within the last decade. On top, recent findings challenge our simplistic view of the cell death signaling machinery as a sole executer of cellular demise, as emerging evidences suggest that some of the classic cell death regulators even promote blood vessel formation. This review summarizes our current knowledge on the role of the cell death signaling machinery with a focus on the apoptosis and necroptosis signaling pathways during blood vessel formation in development and pathology.

Neuronal LRP4 regulates synapse formation in the developing CNS.

The low-density lipoprotein receptor-related protein 4 (LRP4) is essential in muscle fibers for the establishment of the neuromuscular junction. Here, we show that LRP4 is also expressed by embryonic cortical and hippocampal neurons, and that downregulation of LRP4 in these neurons causes a reduction in density of synapses and number of primary dendrites. Accordingly, overexpression of LRP4 in cultured neurons had the opposite effect inducing more but shorter primary dendrites with an increased number of spines. Transsynaptic tracing mediated by rabies virus revealed a reduced number of neurons presynaptic to the cortical neurons in which LRP4 was knocked down. Moreover, neuron-specific knockdown of LRP4 by in utero electroporation of LRP4 miRNA in vivo also resulted in neurons with fewer primary dendrites and a lower density of spines in the developing cortex and hippocampus. Collectively, our results demonstrate an essential and novel role of neuronal LRP4 in dendritic development and synaptogenesis in the CNS.

Risk and protective factors for anxiety and obsessive-compulsive disorders: an umbrella review of systematic reviews and meta-analyses.

BACKGROUND: A multitude of risk/protective factors for anxiety and obsessive-compulsive disorders have been proposed. We conducted an umbrella review to summarize the evidence of the associations between risk/protective factors and each of the following disorders: specific phobia, social anxiety disorder, generalized anxiety disorder, panic disorder, and obsessive-compulsive disorder, and to assess the strength of this evidence whilst controlling for several biases. METHODS: Publication databases were searched for systematic reviews and meta-analyses examining associations between potential risk/protective factors and each of the disorders investigated. The evidence of the association between each factor and disorder was graded into convincing, highly suggestive, suggestive, weak, or non-significant according to a standardized classification based on: number of cases (>1000), random-effects p-values, 95% prediction intervals, confidence interval of the largest study, heterogeneity between studies, study effects, and excess of significance. RESULTS: Nineteen systematic reviews and meta-analyses were included, corresponding to 216 individual studies covering 427 potential risk/protective factors. Only one factor association (early physical trauma as a risk factor for social anxiety disorder, OR 2.59, 95% CI 2.17-3.1) met all the criteria for convincing evidence. When excluding the requirement for more than 1000 cases, five factor associations met the other criteria for convincing evidence and 22 met the remaining criteria for highly suggestive evidence. CONCLUSIONS: Although the amount and quality of the evidence for most risk/protective factors for anxiety and obsessive-compulsive disorders is limited, a number of factors significantly increase the risk for these disorders, may have potential prognostic ability and inform prevention.

Analysis of KCNH2 and CACNA1C schizophrenia risk genes on EEG functional network modulation during an auditory odd-ball task.

A deficit in task-related functional connectivity modulation from electroencephalogram (EEG) has been described in schizophrenia. The use of measures of neuronal connectivity as an intermediate phenotype may allow identifying genetic factors involved in these deficits, and therefore, establishing underlying pathophysiological mechanisms. Genes involved in neuronal excitability and previously associated with the risk for schizophrenia may be adequate candidates in relation to functional connectivity alterations in schizophrenia. The objective was to study the association of two genes of voltage-gated ion channels (CACNA1C and KCNH2) with the functional modulation of the cortical networks measured with EEG and graph-theory parameter during a cognitive task, both in individuals with schizophrenia and healthy controls. Both CACNA1C (rs1006737) and KCNH2 (rs3800779) were genotyped in 101 controls and 50 schizophrenia patients. Small-world index (SW) was calculated from EEG recorded during an odd-ball task in two different temporal windows (pre-stimulus and response). Modulation was defined as the difference in SW between both windows. Genetic, group and their interaction effects on SW in the pre-stimulus window and in modulation were evaluated using ANOVA. The CACNA1C genotype was not associated with SW properties. KCNH2 was significantly associated with SW modulation. Healthy subjects showed a positive SW modulation irrespective of the KCNH2 genotype, whereas within patients allele-related differences were observed. Patients carrying the KCNH2 risk allele (A) presented a negative SW modulation and non-carriers showed SW modulation similar to the healthy subjects. Our data suggest that KCNH2 genotype contributes to the efficient modulation of brain electrophysiological activity during a cognitive task in schizophrenia patients.

Splenectomy reduces lung metastases and tumoral and metastatic niche inflammation.

The immune microenvironment plays a crucial role in supporting tumor growth and metastasis. Tumor-associated macrophages (TAMs) and neutrophils (TANs) are essential components of this microenvironment and affect tumor growth and progression in almost all solid neoplasms. Furthermore, TAMs, TANs and tumor-infiltrating dendritic cells (TIDCs) are found to infiltrate specific distant organs to prepare them as a site for metastatic cell seeding, forming the pre-metastatic niche. The spleen was identified as a major reservoir and source of circulating and tumor infiltrating immune cells. However, discrepancies about its role in supporting tumor growth exist. Thus, here we investigated the role of splenectomy in primary tumor and metastatic growth, and in the formation of an inflammatory niche. In a murine 4T1 and E0771 breast and Panc02 pancreatic cancer model, our results show that while splenectomy reduces the number of infiltrating TAMs, TANs and TIDCs within primary tumors, it does not affect its growth. In line, fewer TAMs, TANs and TIDCs accumulate in the metastatic microenvironment after splenectomy. Interestingly though, this affected metastatic growth depending on the metastatic route/site. The number of hematogenous breast cancer lung metastases was reduced after splenectomy but no effect was observed in breast or pancreatic lymph node metastases. Moreover, we observed that the immune composition of the pre-metastatic niche in lungs of breast cancer bearing mice was altered, and that this could cause the reduction of metastases. Altogether, our results highlight that splenectomy affects the immune microenvironment not only of primary tumors but also of pre-metastatic and metastatic sites.

Role of retinal pigment epithelium-derived exosomes and autophagy in new blood vessel formation.

Autophagy and exosome secretion play important roles in a variety of physiological and disease states, including the development of age-related macular degeneration. Previous studies have demonstrated that these cellular mechanisms share common pathways of activation. Low oxidative damage in ARPE-19 cells, alters both autophagy and exosome biogenesis. Moreover, oxidative stress modifies the protein and genetic cargo of exosomes, possibly affecting the fate of surrounding cells. In order to understand the connection between these two mechanisms and their impact on angiogenesis, stressed ARPE-19 cells were treated with a siRNA-targeting Atg7, a key protein for the formation of autophagosomes. Subsequently, we observed the formation of multivesicular bodies and the release of exosomes. Released exosomes contained VEGFR2 as part of their cargo. This receptor for VEGF-which is critical for the development of new blood vessels-was higher in exosome populations released from stressed ARPE-19. While stressed exosomes enhanced tube formation, exosomes became ineffective after silencing VEGFR2 in ARPE-19 cells and were, consequently, unable to influence angiogenesis. Moreover, vessel sprouting in the presence of stressed exosomes seems to follow a VEGF-independent pathway. We propose that abnormal vessel growth correlates with VEGFR2-expressing exosomes release from stressed ARPE-19 cells, and is directly linked to autophagy.

A PRDX1-p38α heterodimer amplifies MET-driven invasion of IDH-wildtype and IDH-mutant gliomas.

The Peroxiredoxin 1 (PRDX1) gene maps to chromosome arm 1p and is hemizygously deleted and epigenetically silenced in isocitrate dehydrogenase 1 or 2 (IDH)-mutant and 1p/19q-codeleted oligodendroglial tumors. In contrast, IDH-wildtype astrocytic gliomas including glioblastomas mostly lack epigenetic silencing and express PRDX1 protein. In our study, we investigated how PRDX1 contributes to the infiltrative growth of IDH-wildtype gliomas. Focusing on p38α-dependent pathways, we analyzed clinical data from 133 patients of the NOA-04 trial cohort to look for differences in the gene expression profiles of gliomas with wildtype or mutant IDH. Biochemical interaction studies as well as in vitro and ex vivo migration studies were used to establish a biological role of PRDX1 in maintaining pathway activity. Whole-brain high-resolution ultramicroscopy and survival analyses of pre-clinical mouse models for IDH-wildtype gliomas were then used for in vivo confirmation. Based on clinical data, we found that the absence of PRDX1 is associated with changes in the expression of MET/HGF signaling components. PRDX1 forms a heterodimer with p38α mitogen-activated protein kinase 14 (MAPK14), stabilizing phospho-p38α in glioma cells. This process amplifies hepatocyte growth factor (HGF)-mediated signaling and stimulates actin cytoskeleton dynamics that promote glioma cell migration. Whole-brain high-resolution ultramicroscopy confirms these findings, indicating that PRDX1 promotes glioma brain invasion in vivo. Finally, reduced expression of PRDX1 increased survival in mouse glioma models. Thus, our preclinical findings suggest that PRDX1 expression levels may serve as a molecular marker for patients who could benefit from targeted inhibition of MET/HGF signaling.

VEGF ligands and receptors: implications in neurodevelopment and neurodegeneration.

Intensive research in the last decade shows that the prototypic angiogenic factor vascular endothelial growth factor (VEGF) can have direct effects in neurons and modulate processes such as neuronal migration, axon outgrowth, axon guidance and neuronal survival. Depending on the neuronal cell type and the process, VEGF seems to exert these effects by signaling via different receptors. It is also becoming clear that other VEGF ligands such as VEGF-B, -C and -D can act in various neuronal cell types as well. Moreover, apart from playing a role in physiological conditions, VEGF and VEGF-B have been related to different neurological disorders. We give an update on how VEGF controls different processes during neurodevelopment as well as on its role in several neurodegenerative disorders. We also discuss recent findings demonstrating that other VEGF ligands influence processes such as neurogenesis and dendrite arborization and participate in neurodegeneration.

VEGF modulates NMDA receptors activity in cerebellar granule cells through Src-family kinases before synapse formation.

NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca(2+) influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca(2+) influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).

Local cannabinoid receptor type-1 regulates glial cell activity and insulin-like growth factor-1 receptor signaling in the mediobasal hypothalamus.

As organisms age, the activity of the endocannabinoid system in the brain declines, coinciding with increased neuroinflammation and disrupted hypothalamic functions. Notably, cannabinoid receptors type-1 (CB1) are highly expressed in the ventromedial hypothalamic nucleus (VMH) within the mediobasal hypothalamus, a central area of neuroendocrine regulation. This study investigates whether the CB1 receptor influences age-related changes in a brain region-dependent manner. Therefore, we performed stereotaxic injections of rAAV1/2 expressing Cre recombinase in 2-month-old CB1flox/flox male animals to delete the CB1 gene and in CB1-deficient (CB1-STOP) mice to induce its re-expression. The intensity of pro-inflammatory glial activity, gonadotropin-releasing hormone (GnRH) and insulin-like growth factor-1 receptor (IGF-1R) expression was assessed in the hypothalamus of mice at 18-19 months of age. Site-specific CB1 receptor deletion induced pro-inflammatory glial activity and increased hypothalamic Igf1r mRNA expression. Unexpectedly, GnRH levels remained unaltered. Importantly, rescuing the receptor in null mutant animals had the opposite effect: it reduced pro-inflammatory glial activation and decreased Igf1r mRNA expression without affecting GnRH production. Overall, the study highlights the important role of the CB1 receptor in the VMH in reducing age-related inflammation and modulating IGF-1R signaling.

The NADPH oxidase Nox2 regulates VEGFR1/CSF-1R-mediated microglial chemotaxis and promotes early postnatal infiltration of phagocytes in the subventricular zone of the mouse cerebral cortex.

The phagocyte NADPH oxidase Nox2 generates superoxide ions implicated in the elimination of microorganisms and the redox control of inflammatory signaling. However, the role of Nox2 in phagocyte functions unrelated to immunity or pathologies is unknown. During development, oriented cell migrations insure the timely recruitment and function of phagocytes in developing tissues. Here, we have addressed the role of Nox2 in the directional migration of microglial cells during development. We show that microglial Nox2 regulates the chemotaxis of purified microglia mediated by the colony stimulating factor-1 receptor (CSF-1R) and the vascular endothelial growth factor receptor-1 (VEGFR1). Stimulation of these receptors triggers activation of Nox2 at the leading edge of polarized cells. In the early postnatal stages of mouse brain development, Nox2 is activated in macrophages / microglial cells in the lateral ventricle or the adjacent subventricular zone (SVZ). Fluorescent microglia injected into the lateral ventricle infiltrate the dorso-caudal SVZ through a mechanism that is blocked by pretreatment of the injected cells with an irreversible Nox inhibitor. Infiltration of endogenous microglia into the caudal SVZ of the cerebral cortex is prevented by (1) Nox2 gene deficiency, (2) treatment with a Nox2 inhibitor (apocynin), and (3) invalidation of the VEGFR1 kinase. We conclude that phagocytes move out of the lateral ventricle soon after birth and infiltrate the cortical SVZ through a mechanism requiring microglial Nox2 and VEGFR1 activation. Nox2 therefore modulates the migration of microglia and their development.

The vasculature of neurogenic niches: Properties and function.

In the adult rodent brain, neural stem cells (NSCs) reside in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. In these areas, NSCs and their progeny integrate intrinsic signals and extrinsic cues provided by their microenvironment that control their behavior. The vasculature in the SVZ and SGZ, and the choroid plexus (ChP) in the SVZ, have emerged as critical compartments of the neurogenic niches as they provide a rich repertoire of cues to regulate NSC quiescence, proliferation, self-renewal and differentiation. Physical contact between NSCs and blood vessels is also a feature within the niches and supports different processes such as quiescence, migration and vesicle transport. In this review, we provide a description of the brain and choroid plexus vasculature in both stem cell niches, highlighting the main properties and role of the vasculature in each niche. We also summarize the current understanding of how blood vessel- and ChP-derived signals influence the behavior of NSCs in physiological adulthood, as well as upon aging.

Endothelial PlexinD1 signaling instructs spinal cord vascularization and motor neuron development.

How the vascular and neural compartment cooperate to achieve such a complex and highly specialized structure as the central nervous system is still unclear. Here, we reveal a crosstalk between motor neurons (MNs) and endothelial cells (ECs), necessary for the coordinated development of MNs. By analyzing cell-to-cell interaction profiles of the mouse developing spinal cord, we uncovered semaphorin 3C (Sema3C) and PlexinD1 as a communication axis between MNs and ECs. Using cell-specific knockout mice and in vitro assays, we demonstrate that removal of Sema3C in MNs, or its receptor PlexinD1 in ECs, results in premature and aberrant vascularization of MN columns. Those vascular defects impair MN axon exit from the spinal cord. Impaired PlexinD1 signaling in ECs also causes MN maturation defects at later stages. This study highlights the importance of a timely and spatially controlled communication between MNs and ECs for proper spinal cord development.

Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice.

The gut has a specific vascular barrier that controls trafficking of antigens and microbiota into the bloodstream. However, the molecular mechanisms regulating the maintenance of this vascular barrier remain elusive. Here, we identified Caspase-8 as a pro-survival factor in mature intestinal endothelial cells that is required to actively maintain vascular homeostasis in the small intestine in an organ-specific manner. In particular, we find that deletion of Caspase-8 in endothelial cells results in small intestinal hemorrhages and bowel inflammation, while all other organs remained unaffected. We also show that Caspase-8 seems to be particularly needed in lymphatic endothelial cells to maintain gut homeostasis. Our work demonstrates that endothelial cell dysfunction, leading to the breakdown of the gut-vascular barrier, is an active driver of chronic small intestinal inflammation, highlighting the role of the intestinal vasculature as a safeguard of organ function.

Matrix-binding vascular endothelial growth factor (VEGF) isoforms guide granule cell migration in the cerebellum via VEGF receptor Flk1.

Vascular endothelial growth factor (VEGF) regulates angiogenesis, but also has important, yet poorly characterized roles in neuronal wiring. Using several genetic and in vitro approaches, we discovered a novel role for VEGF in the control of cerebellar granule cell (GC) migration from the external granule cell layer (EGL) toward the Purkinje cell layer (PCL). GCs express the VEGF receptor Flk1, and are chemoattracted by VEGF, whose levels are higher in the PCL than EGL. Lowering VEGF levels in mice in vivo or ectopic VEGF expression in the EGL ex vivo perturbs GC migration. Using GC-specific Flk1 knock-out mice, we provide for the first time in vivo evidence for a direct chemoattractive effect of VEGF on neurons via Flk1 signaling. Finally, using knock-in mice expressing single VEGF isoforms, we show that pericellular deposition of matrix-bound VEGF isoforms around PC dendrites is necessary for proper GC migration in vivo. These findings identify a previously unknown role for VEGF in neuronal migration.

Epithelial: Endothelial cross-talk regulates exocrine differentiation in developing pancreas.

Endothelial cells are required to initiate pancreas development from the endoderm. They also control the function of endocrine islets after birth. Here we investigate in developing pancreas how the endothelial cells become organized during branching morphogenesis and how their development affects pancreatic cell differentiation. We show that endothelial cells closely surround the epithelial bud at the onset of pancreas morphogenesis. During branching morphogenesis, the endothelial cells become preferentially located near the central (trunk) epithelial cells and remain at a distance from the branch tips where acinar cells differentiate. This correlates with predominant expression of the angiogenic factor vascular endothelial growth factor-A (VEGF-A) in trunk cells. In vivo ablation of VEGF-A expression by pancreas-specific inactivation of floxed Vegfa alleles results in reduced endothelial development and in excessive acinar differentiation. On the contrary, acinar differentiation is repressed when endothelial cells are recruited around tip cells that overexpress VEGF-A. Treatment of embryonic day 12.5 explants with VEGF-A or with VEGF receptor antagonists confirms that acinar development is tightly controlled by endothelial cells. We also provide evidence that endothelial cells repress the expression of Ptf1a, a transcription factor essential for acinar differentiation, and stimulate the expression of Hey-1 and Hey-2, two repressors of Ptf1a activity. In explants, we provide evidence that VEGF-A signaling is required, but not sufficient, to induce endocrine differentiation. In conclusion, our data suggest that, in developing pancreas, epithelial production of VEGF-A determines the spatial organization of endothelial cells which, in turn, limit acinar differentiation of the epithelium.

Impaired autonomic regulation of resistance arteries in mice with low vascular endothelial growth factor or upon vascular endothelial growth factor trap delivery.

BACKGROUND: Control of peripheral resistance arteries by autonomic nerves is essential for the regulation of blood flow. The signals responsible for the maintenance of vascular neuroeffector mechanisms in the adult, however, remain largely unknown. METHODS AND RESULTS: Here, we report that VEGF( partial differential/ partial differential) mice with low vascular endothelial growth factor (VEGF) levels suffer defects in the regulation of resistance arteries. These defects are due to dysfunction and structural remodeling of the neuroeffector junction, the equivalent of a synapse between autonomic nerve endings and vascular smooth muscle cells, and to an impaired contractile smooth muscle cell phenotype. Notably, short-term delivery of a VEGF inhibitor to healthy mice also resulted in functional and structural defects of neuroeffector junctions. CONCLUSIONS: These findings uncover a novel role for VEGF in the maintenance of arterial neuroeffector function and may help us better understand how VEGF inhibitors cause vascular regulation defects in cancer patients.