Marta Segarra

Elevated Production of Interleukin-6 Is Associated With a Lower Incidence of Disease-Related Ischemic Events in Patients With Giant-Cell Arteritis. Angiogenic Activity of Interleukin-6 as a Potential Protective Mechanism

Background—Patients with giant-cell arteritis (GCA) who develop a strong acute-phase response are at low risk of disease-related ischemic events. Methods and Results—To assess the potential protective role of proinflammatory cytokines in the development of ischemic events in GCA, we measured tissue expression (66 individuals) and/or circulating levels (80 individuals) of interleukin (IL)-1&bgr;, tumor necrosis factor-&agr; (TNF-&agr;), and IL-6 in patients with biopsy-proven GCA. Tissue expression was determined by quantitative real-time polymerase chain reaction and immunohistochemistry. Circulating cytokines were determined by enzyme-linked immunoassay. We found that patients with disease-related ischemic events had lower IL-6 mRNA levels (5.9±2.1 versus 27.6±7.8 relative units, P =0.013), lower IL-6 immunohistochemical expression scores (1.5±0.9 versus 2.7±1, P =0.001), and lower circulating levels of IL-6 (13.6±2.1 versus 24±2.4 pg/mL, P =0.002) than patients without ischemic complications. No significant differences were found for either IL-1&bgr; or TNF-&agr;. We subsequently investigated direct effects of IL-6 on vessel wall components. We found that IL-6 stimulates endothelial cell proliferation and differentiation into capillary-like structures and induces full angiogenic activity in both ex vivo (aortic ring) and in vivo (chick chorioallantoic membrane) assays. Conclusions—GCA patients with ischemic complications have lower tissue expression and circulating levels of IL-6 than patients with no ischemic events. IL-6 has relevant direct effects on vascular wall components that might be protective: IL-6 activates a functional program related to angiogenesis that may compensate for ischemia in patients with GCA.

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.

Impaired Recruitment of Grk6 and β-Arrestin2 Causes Delayed Internalization and Desensitization of a WHIM Syndrome-Associated CXCR4 Mutant Receptor

WHIM (warts, hypogammaglobulinemia, infections, and myelokatexis) syndrome is a rare immunodeficiency syndrome linked to heterozygous mutations of the chemokine receptor CXCR4 resulting in truncations of its cytoplasmic tail. Leukocytes from patients with WHIM syndrome display impaired CXCR4 internalization and enhanced chemotaxis in response to its unique ligand SDF-1/CXCL12, which likely contribute to the clinical manifestations. Here, we investigated the biochemical mechanisms underlying CXCR4 deficiency in WHIM syndrome. We report that after ligand activation, WHIM-associated mutant CXCR4 receptors lacking the carboxy-terminal 19 residues internalize and activate Erk 1/2 slower than wild-type (WT) receptors, while utilizing the same trafficking endocytic pathway. Recruitment of β-Arrestin 2, but not β-Arrestin 1, to the active WHIM-mutant receptor is delayed compared to the WT CXCR4 receptor. In addition, while both kinases Grk3 and Grk6 bind to WT CXCR4 and are critical to its trafficking to the lysosomes, Grk6 fails to associate with the WHIM-mutant receptor whereas Grk3 associates normally. Since β-Arrestins and Grks play critical roles in phosphorylation and internalization of agonist-activated G protein-coupled receptors, these results provide a molecular basis for CXCR4 dysfunction in WHIM syndrome.

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.

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.

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 signaling repressing JNK3 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 signaling is essential for vessel pruning, and that defects in this pathway may contribute to PHPV.

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.

Neuropilin-2: A New Molecular Target for Antiangiogenic and Antitumor Strategies

Aggressive cancers grow progressively, invade locally, and metas-tasize through a multistep process that involves a malignant cell and a supportive environment (1). Tumor neovascularization critically contributes to tumor growth (2). Hence, there has been great interest in reducing cancer progression by shutting down the tumor blood supply (3). The antiangiogenic drugs currently approved for cancer treatment include bevacizumab (Avastin), a humanized monoclonal antibody blocking vascular endothelial growth factor (VEGF), and sorafenib (Nexavar) and sunitinib (Sutent), synthetic inhibitors of VEGF receptor signaling (4 , 5). These VEGF-targeted therapies have shown benefi t in certain cancer types, but responses have generally been modest and measured in months of extended cancer survival (6 – 8). This experience raises many questions regarding the appropriate role of current antiangiogenic therapies in cancer treatment (9). In this issue of the Journal, Gray et al. (10) present exciting results that support a potential new strategy, targeting neuropilin-2. Neuropilins are nonsignaling transmembrane receptors that are shared by two distinct ligand families, the class 3 semaphorins, which regulate neuronal guidance, and the VEGF proteins, which regulate angiogenesis (11 , 12). Ligand binding to neuropilins dictates their association with specifi c signal transducers: semaphorins mediate interactions with plexin A receptors, whereas VEGFs mediate interactions with VEGF receptors. VEGF and Sema3A (one of the semaphorins) have overlapping binding domains on the extracellular portion of neuropilin-1 and, for this reason, can compete with one another for binding (13). Two neuropilins are known, neuropilin-1 and neuropilin-2 that have similar structures but differ in their endothelial expression during development. Neuropilin-1 is expressed mainly in the arterial endothelium, whereas neuropi-lin-2 is expressed mainly in the venous and lymphatic endothelium (14 , 15). Neuropilin-1 interacts with heparin-binding isoforms of VEGF-A,-B,-E and placental growth factor; neuropilin-2 interacts with VEGF-A,-C, and-D (11 , 12). Gene-targeting studies have demonstrated that mice lacking functional neuropilin-1 receptors die during embryogenesis with heart and vascular defects, demonstrating a critical role of neuropilin-1 in vascular development (16 – 18); mice lacking both neuropilin-1 and neuropilin-2 are more severely affected than mice lacking only neuropilin-1 (16 – 18). Neuropilin-2 – defi cient mice develop normally, but they are smaller than wild-type mice and display minor abnormalities in the lym-phatic system (15). Interestingly, neuropilin-2 – defi cient mice also display defective retinal neovascularization in response to ischemia (19). Recent studies have shown that administration of neuropilin-1 – neutralizing antibodies to tumor-bearing mice reduced tumor angiogenesis …

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.

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

Summary 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.