Differential effects of the N-terminal helix of FGF8b on the activity of a small-molecule FGFR inhibitor in cell culture and for the extracellular domain of FGFR3c in solution.

SSR128129E (SSR) is a unique small-molecule inhibitor of fibroblast growth factor receptors (FGFRs). SSR is a high-affinity allosteric binder that selectively blocks one of the two major FGFR-mediated pathways. The mechanisms of SSR activity were studied previously in much detail, allowing the identification of its binding site, located in the hydrophobic groove of the receptor D3 domain. The binding site overlaps with the position of an N-terminal helix, an element exclusive for the FGF8b growth factor, which could potentially convert SSR from an allosteric inhibitor into an orthosteric blocker for the particular FGFR/FGF8b system. In this regard, we report here on the structural and functional investigation of FGF8b/FGFR3c system and the effects imposed on it by SSR. We show that SSR is equally or more potent in inhibiting FGF8b-induced FGFR signaling compared to FGF2-induced activation. On the other hand, when studied in the context of separate extracellular domains of FGFR3c in solution with NMR spectroscopy, SSR is unable to displace the N-terminal helix of FGF8b from its binding site on FGFR3c and behaves as a weak orthosteric inhibitor. The substantial inconsistency between the results obtained with cell culture and for the individual water-soluble subdomains of the FGFR proteins points to the important role played by the cell membrane.

Vascular Regulation of Developmental Neurogenesis.

Evolutionary studies indicate that the nervous system evolved prior to the vascular system, but the increasing complexity of organisms prompted the vascular system to emerge in order to meet the growing demand for oxygen and nutrient supply. In recent years, it has become apparent that the symbiotic communication between the nervous and the vascular systems goes beyond the exclusive covering of the demands on nutrients and oxygen carried by blood vessels. Indeed, this active interplay between both systems is crucial during the development of the central nervous system (CNS). Several neural-derived signals that initiate and regulate the vascularization of the CNS have been described, however less is known about the vascular signals that orchestrate the development of the CNS cytoarchitecture. Here, we focus on reviewing the effects of blood vessels in the process of neurogenesis during CNS development in vertebrates. In mammals, we describe the spatiotemporal features of vascular-driven neurogenesis in two brain regions that exhibit different neurogenic complexity in their germinal zone, the hindbrain and the forebrain.

Cryptosporidium parvum outbreak associated with Raccoons at a Wildlife Facility-Virginia, May-June 2019.

Cryptosporidium parvum is a parasitic zoonotic pathogen responsible for diarrheal illness in humans and animals worldwide. We report an investigation of a cryptosporidiosis outbreak in raccoons and wildlife rehabilitation workers at a Virginia facility. Fifteen (31%) of 49 facility personnel experienced symptoms meeting the case definition, including four laboratory-confirmed cases. Seven juvenile raccoons were reported to have diarrhoea; six had laboratory-confirmed cryptosporidiosis. Cryptosporidium parvum of the same molecular subtype (IIaA16G3R2) was identified in two human cases and six raccoons. Raccoon illness preceded human illness by 11 days, suggesting possible zoonotic transmission from raccoons to humans. This appears to be the first report of a human cryptosporidiosis outbreak associated with exposure to raccoons infected with C. parvum. Raccoons might be an under-recognized reservoir for human C. parvum infections. Further study is needed to explore the prevalence of cryptosporidial species in raccoons and their role as a wildlife reservoir.

Neurovascular crosstalk coordinates the central nervous system development.

PURPOSE OF THE REVIEW: The synchronic development of vascular and nervous systems is orchestrated by common molecules that regulate the communication between both systems. The identification of these common guiding cues and the developmental processes regulated by neurovascular communication are slowly emerging. In this review, we describe the molecules modulating the neurovascular development and their impact in processes such as angiogenesis, neurogenesis, neuronal migration, and brain homeostasis. RECENT FINDINGS: Blood vessels not only are involved in nutrient and oxygen supply of the central nervous system (CNS) but also exert instrumental functions controlling developmental neurogenesis, CNS cytoarchitecture, and neuronal plasticity. Conversely, neurons modulate CNS vascularization and brain endothelial properties such as blood-brain barrier and vascular hyperemia. SUMMARY: The integration of the active role of endothelial cells in the development and maintenance of neuronal function is important to obtain a more holistic view of the CNS complexity and also to understand how the vasculature is involved in neuropathological conditions.

Blood-Brain Barrier Dynamics to Maintain Brain Homeostasis.

The blood-brain barrier (BBB) is a dynamic platform for exchange of substances between the blood and the brain parenchyma, and it is an essential functional gatekeeper for the central nervous system (CNS). While it is widely recognized that BBB disruption is a hallmark of several neurovascular pathologies, an aspect of the BBB that has received somewhat less attention is the dynamic modulation of BBB tightness to maintain brain homeostasis in response to extrinsic environmental factors and physiological changes. In this review, we summarize how BBB integrity adjusts in critical stages along the life span, as well as how BBB permeability can be altered by common stressors derived from nutritional habits, environmental factors and psychological stress.

EphrinB2 regulates VEGFR2 during dendritogenesis and hippocampal circuitry development.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that play important roles in the nervous system, although it is still unclear which receptors transduce those signals in neurons. Here, we show that in the developing hippocampus VEGFR2 (also known as KDR or FLK1) is expressed specifically in the CA3 region and it is required for dendritic arborization and spine morphogenesis in hippocampal neurons. Mice lacking VEGFR2 in neurons (Nes-cre Kdrlox/-) show decreased dendritic arbors and spines as well as a reduction in long-term potentiation (LTP) at the associational-commissural - CA3 synapses. Mechanistically, VEGFR2 internalization is required for VEGF-induced spine maturation. In analogy to endothelial cells, ephrinB2 controls VEGFR2 internalization in neurons. VEGFR2-ephrinB2 compound mice (Nes-cre Kdrlox/+ Efnb2lox/+) show reduced dendritic branching, reduced spine head size and impaired LTP. Our results demonstrate the functional crosstalk of VEGFR2 and ephrinB2 in vivo to control dendritic arborization, spine morphogenesis and hippocampal circuitry development.

Neurovascular Interactions in the Nervous System.

Molecular cross talk between the nervous and vascular systems is necessary to maintain the correct coupling of organ structure and function. Molecular pathways shared by both systems are emerging as major players in the communication of the neuronal compartment with the endothelium. Here we review different aspects of this cross talk and how vessels influence the development and homeostasis of the nervous system. Beyond the classical role of the vasculature as a conduit to deliver oxygen and metabolites needed for the energy-demanding neuronal compartment, vessels emerge as powerful signaling systems that control and instruct a variety of cellular processes during the development of neurons and glia, such as migration, differentiation, and structural connectivity. Moreover, a broad spectrum of mild to severe vascular dysfunctions occur in various pathologies of the nervous system, suggesting that mild structural and functional changes at the neurovascular interface may underlie cognitive decline in many of these pathological conditions.

Endothelial Dab1 signaling orchestrates neuro-glia-vessel communication in the central nervous system.

The architecture of the neurovascular unit (NVU) is controlled by the communication of neurons, glia, and vascular cells. We found that the neuronal guidance cue reelin possesses proangiogenic activities that ensure the communication of endothelial cells (ECs) with the glia to control neuronal migration and the establishment of the blood-brain barrier in the mouse brain. Apolipoprotein E receptor 2 (ApoER2) and Disabled1 (Dab1) expressed in ECs are required for vascularization of the retina and the cerebral cortex. Deletion of Dab1 in ECs leads to a reduced secretion of laminin-α4 and decreased activation of integrin-ß1 in glial cells, which in turn control neuronal migration and barrier properties of the NVU. Thus, reelin signaling in the endothelium is an instructive and integrative cue essential for neuro-glia-vascular communication.

GRIP1 Binds to ApoER2 and EphrinB2 to Induce Activity-Dependent AMPA Receptor Insertion at the Synapse.

Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in response to neuronal activity is critical for synaptic function and plasticity. Here, we show that neuronal activity induces the binding of ephrinB2 and ApoER2 receptors at the postsynapse to regulate de novo insertion of AMPA receptors. Mechanistically, the multi-PDZ adaptor glutamate-receptor-interacting protein 1 (GRIP1) binds ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors. Phosphorylation of ephrinB2 in a serine residue (Ser-9) is essential for the stability of such a complex. In vivo, a mutation on ephrinB2 Ser-9 in mice results in a complete disruption of the complex, absence of ApoER2 downstream signaling, and impaired activity-induced and ApoER2-mediated AMPA receptor insertion. Using compound genetics, we show the requirement of this complex for long-term potentiation (LTP). Together, our findings uncover a cooperative ephrinB2 and ApoER2 signaling at the synapse, which serves to modulate activity-dependent AMPA receptor dynamic changes during synaptic plasticity.

Endothelin-1 promotes vascular smooth muscle cell migration across the artery wall: a mechanism contributing to vascular remodelling and intimal hyperplasia in giant-cell arteritis.

BACKGROUND: Giant-cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries, frequently involving the temporal arteries (TA). Inflammation-induced vascular remodelling leads to vaso-occlusive events. Circulating endothelin-1 (ET-1) is increased in patients with GCA with ischaemic complications suggesting a role for ET-1 in vascular occlusion beyond its vasoactive function. OBJECTIVE: To investigate whether ET-1 induces a migratory myofibroblastic phenotype in human TA-derived vascular smooth muscle cells (VSMC) leading to intimal hyperplasia and vascular occlusion in GCA. METHODS AND RESULTS: Immunofluorescence/confocal microscopy showed increased ET-1 expression in GCA lesions compared with control arteries. In inflamed arteries, ET-1 was predominantly expressed by infiltrating mononuclear cells whereas ET receptors, particularly ET-1 receptor B (ETBR), were expressed by both mononuclear cells and VSMC. ET-1 increased TA-derived VSMC migration in vitro and α-smooth muscle actin (αSMA) expression and migration from the media to the intima in cultured TA explants. ET-1 promoted VSMC motility by increasing activation of focal adhesion kinase (FAK), a crucial molecule in the turnover of focal adhesions during cell migration. FAK activation resulted in Y397 autophosphorylation creating binding sites for Src kinases and the p85 subunit of PI3kinases which, upon ET-1 exposure, colocalised with FAK at the focal adhesions of migrating VSMC. Accordingly, FAK or PI3K inhibition abrogated ET-1-induced migration in vitro. Consistently, ET-1 receptor A and ETBR antagonists reduced αSMA expression and delayed VSMC outgrowth from cultured GCA-involved artery explants. CONCLUSIONS: ET-1 is upregulated in GCA lesions and, by promoting VSMC migration towards the intimal layer, may contribute to intimal hyperplasia and vascular occlusion in GCA.

A vascular perspective on neuronal migration.

During CNS development and adult neurogenesis, immature neurons travel from the germinal zones towards their final destination using cellular substrates for their migration. Classically, radial glia and neuronal axons have been shown to act as physical scaffolds to support neuroblast locomotion in processes known as gliophilic and neurophilic migration, respectively (Hatten, 1999; Marin and Rubenstein, 2003; Rakic, 2003). In adulthood, long distance neuronal migration occurs in a glial-independent manner since radial glia cells differentiate into astrocytes after birth. A series of studies highlight a novel mode of neuronal migration that uses blood vessels as scaffolds, the so-called vasophilic migration. This migration mode allows neuroblast navigation in physiological and also pathological conditions, such as neuronal precursor migration after ischemic stroke or cerebral invasion of glioma tumor cells. Here we review the current knowledge about how vessels pave the path for migrating neurons and how trophic factors derived by glio-vascular structures guide neuronal migration both during physiological as well as pathological processes.

EphrinB2 controls vessel pruning through STAT1-JNK3 signalling.

Angiogenesis produces primitive vascular networks that need pruning to yield hierarchically organized and functional vessels. Despite the critical importance of vessel pruning to vessel patterning and function, the mechanisms regulating this process are not clear. Here we show that EphrinB2, a well-known player in angiogenesis, is an essential regulator of endothelial cell death and vessel pruning. This regulation depends upon phosphotyrosine-EphrinB2 signalling repressing c-jun N-terminal kinase 3 activity via STAT1. JNK3 activation causes endothelial cell death. In the absence of JNK3, hyaloid vessel physiological pruning is impaired, associated with abnormal persistence of hyaloid vessels, defective retinal vasculature and microphthalmia. This syndrome closely resembles human persistent hyperplastic primary vitreus (PHPV), attributed to failed involution of hyaloid vessels. Our results provide evidence that EphrinB2/STAT1/JNK3 signalling is essential for vessel pruning, and that defects in this pathway may contribute to PHPV.

Increased IL-17A expression in temporal artery lesions is a predictor of sustained response to glucocorticoid treatment in patients with giant-cell arteritis.

BACKGROUND: Interleukin 17A (IL-17A) exerts pivotal proinflammatory functions in chronic inflammatory and autoimmune diseases. OBJECTIVE: To investigate IL-17A expression in temporal artery lesions from patients with giant-cell arteritis (GCA), and its relationship with disease outcome. METHODS: Fifty-seven patients with biopsy-proven GCA were prospectively evaluated, treated and followed for 4.5 years (52-464 weeks). Relapses, time (weeks) required to achieve a maintenance prednisone dose <10 mg/day, and time (weeks) to complete prednisone withdrawal were prospectively recorded. IL-17A mRNA was measured by real-time quantitative RT-PCR in temporal arteries from all patients and 19 controls. IL-17 protein expression was assessed by immunohistochemistry/immunofluorescence. RESULTS: IL-17A expression was significantly increased in temporal artery samples from GCA patients compared with controls (6.22±8.61 vs 2.50±3.9 relative units, p=0.016). Surprisingly, patients with strong IL-17A expression tended to experience less relapses, and required significantly shorter treatment periods (median 25 vs 44 weeks to achieve <10 mg prednisone/day, p=0.0079). There was no correlation between IL-17A and RORc or RORα expression suggesting that these transcription factors may not exclusively reflect Th17 differentiation, and that cells other than Th17 cells might contribute to IL-17 expression in active patients. Accordingly, FoxP3(+)IL-17A(+) cells were identified in lesions by confocal microscopy and were dramatically reduced in specimens from treated patients. CONCLUSIONS: IL-17A expression is increased in GCA lesions, and is a predictor of response to glucocorticoid treatment. The contribution of FoxP3+ cells to IL-17A production in untreated patients suggests that induced-Tregs may facilitate disease remission when proinflammatory cytokine production is downregulated by glucocorticosteroids.

Semaphorin 6A regulates angiogenesis by modulating VEGF signaling.

Formation of new vessels during development and in the mature mammal generally proceeds through angiogenesis. Although a variety of molecules and signaling pathways are known to underlie endothelial cell sprouting and remodeling during angiogenesis, many aspects of this complex process remain unexplained. Here we show that the transmembrane semaphorin6A (Sema6A) is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating the expression and signaling of VEGFR2, which is known to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of prosurvival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, matrigel, and choroidal angiogenesis compared with controls. Sema6A plays important roles in development of the nervous system. Here we show that it also regulates vascular development and adult angiogenesis.

Tissue and serum markers of inflammation during the follow-up of patients with giant-cell arteritis--a prospective longitudinal study.

OBJECTIVE: To evaluate the association between inflammatory markers and relapse in GCA patients longitudinally assessed in a clinical trial of infliximab and glucocorticosteroids. METHODS: Forty-four newly diagnosed GCA patients in glucocorticosteroid-induced remission were randomized to receive infliximab 5 mg/kg or placebo plus daily glucocorticosteroids, tapered using a standardized schedule. Sera were analysed for inflammatory markers at multiple, pre-defined time points. Temporal artery biopsies were performed in four patients before and after treatment to analyse changes in inflammatory and vascular remodelling marker expression. RESULTS: Thirteen of 44 patients relapsed. Similar proportions of relapsed patients were present in both treatment arms. ESR, CRP, intercellular adhesion molecule (ICAM)-1, TNF-α, and IL-12p40 were significantly elevated near relapse. In post-treatment biopsies, mRNA expression of pro-inflammatory cytokines decreased, while vascular remodelling factors increased relative to baseline biopsies. Tissue IL-12p40 and IFN-γ mRNA remained elevated in relapsing vs remitting patients. CONCLUSION: Despite prior findings of high concentrations of TNF-α in temporal artery biopsies of GCA patients, infliximab plus glucocorticosteroids did not result in improved clinical outcomes. Increased measures of this biomarker did not provide useful insight into the relative importance of TNF-α in the pathogenesis of GCA. Gene expression analysis in paired temporal artery biopsies pre- and post-treatment revealed decreased inflammatory activity and active vascular remodelling following treatment. In relapsing patients, increased expression of IFN-γ and IL-12p40 in post-treatment biopsies suggests a role in sustaining disease and setting the stage for relapse during treatment withdrawal. TRIAL REGISTRATION: ClinicalTrials.gov; http://www.clinicaltrials.gov; NCT00076726.

Adult human circulating CD34⁻Lin⁻CD45⁻CD133⁻ cells can differentiate into hematopoietic and endothelial cells.

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

Oligo-guanosine nucleotide induces neuropilin-1 internalization in endothelial cells and inhibits angiogenesis.

Ligand interaction with cognate cell-surface receptor often promotes receptor internalization, protecting cells from prolonged or excessive signaling from extracellular ligands. Compounds that induce internalization of surface receptors prevent ligand binding to cognate cell-surface receptors serving as inhibitors. Here, we show that synthetic polyriboguanosine (poly G) and oligo-deoxyriboguanosine (oligo G) reduce endothelial levels of surface neuropilin-1 (NRP1), a receptor shared by semaphorin 3A and vascular endothelial growth factor (VEGF), which plays critical roles in angiogenesis. Oligo G also reduces levels of cell-surface scavenger receptor expressed by endothelial cells I (SREC-I), but not levels of NRP2, gp130, CD31, VEGFR-1, or VEGFR-2. Poly or oligo A, T, and C do not promote NRP1 or SREC-I internalization. We find that oligo G binds to NRP1 with high affinity (Kd:1.3 ± 0.16 nM), bridges the extracellular domain of NRP1 to that of SREC-I, and induces coordinate internalization of NRP1 and SREC-I. In vitro, oligo G blocks the binding and function of VEGF(165) in endothelial cells. In vivo, intravitreal administration of oligo G reduces choroidal neovascularization in mice. These results demonstrate that synthetic oligo G is an inhibitor of pathologic angiogenesis that reduces cell-surface levels and function of NRP1 acting as an internalization inducer.

Clinical relevance of persistently elevated circulating cytokines (tumor necrosis factor alpha and interleukin-6) in the long-term followup of patients with giant cell arteritis.

OBJECTIVE: To assess the clinical relevance of increased circulating cytokines in patients with giant cell arteritis (GCA) after long-term followup. METHODS: We performed a cross-sectional evaluation of 54 patients with biopsy-proven GCA prospectively followed for a median of 5.4 years (range 4-10.5 years). GCA-related complications, vascular events, relapses, current prednisone dose, time required to achieve a maintenance prednisone dosage <10 mg/day, cumulated prednisone at that point, and adverse effects during followup were recorded. Serum interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) were determined by immunoassay. RESULTS: All patients were in clinical remission. Both cytokines were significantly higher in patients than in controls (mean +/- SD 21 +/- 35 versus 5 +/- 11 pg/ml; P < 0.001 for IL-6 and mean +/- SD 32 +/- 14 versus 16 +/- 9 pg/ml; P < 0.001 for TNFalpha). No differences were found in patients with or without GCA-related complications or vascular events during followup. Circulating cytokines were significantly higher in patients who had experienced relapses (mean +/- SD 25 +/- 39 versus 10 +/- 11 pg/ml; P = 0.04 for IL-6 and mean +/- SD 34 +/- 15 versus 25 +/- 11 pg/ml; P = 0.042 for TNFalpha). IL-6 was significantly higher in patients still requiring prednisone (mean +/- SD 29 +/- 45 versus 13 +/- 17 pg/ml; P = 0.008), and TNFalpha correlated with cumulated prednisone dose (r = 0.292, P = 0.04). No significant relationship was found between elevated cytokines and prednisone adverse effects or patients' quality of life. CONCLUSION: Circulating TNFalpha and IL-6 may persist elevated in GCA patients after long-term followup and remain higher in patients who have experienced more relapsing disease. However, in this patient cohort, elevated circulating cytokines were not associated with increased frequency of GCA complications, vascular events, or treatment-related side effects.

The transcription factor Gfi1 regulates G-CSF signaling and neutrophil development through the Ras activator RasGRP1.

The transcription factor growth factor independence 1 (Gfi1) and the growth factor granulocyte colony-stimulating factor (G-CSF) are individually essential for neutrophil differentiation from myeloid progenitors. Here, we provide evidence that the functions of Gfi1 and G-CSF are linked in the regulation of granulopoiesis. We report that Gfi1 promotes the expression of Ras guanine nucleotide releasing protein 1 (RasGRP1), an exchange factor that activates Ras, and that RasGRP1 is required for G-CSF signaling through the Ras/mitogen-activated protein/extracellular signal-regulated kinase (MEK/Erk) pathway. Gfi1-null mice have reduced levels of RasGRP1 mRNA and protein in thymus, spleen, and bone marrow, and Gfi1 transduction in myeloid cells promotes RasGRP1 expression. When stimulated with G-CSF, Gfi1-null myeloid cells are selectively defective at activating Erk1/2, but not signal transducer and activator of transcription 1 (STAT1) or STAT3, and fail to differentiate into neutrophils. Expression of RasGRP1 in Gfi1-deficient cells rescues Erk1/2 activation by G-CSF and allows neutrophil maturation by G-CSF. These results uncover a previously unknown function of Gfi1 as a regulator of RasGRP1 and link Gfi1 transcriptional control to G-CSF signaling and regulation of granulopoiesis.