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Neuropilin-2(Nrp2), which is a nontyrosine kinase transmembrane glycoprotein, can promote angiogenesis and is a poor prognostic factor in some human cancers. In the present study, to explore the expression and potential function of Nrp2 in salivary adenoid cystic carcinoma (SACC), immunohistochemistry was used to examine the Nrp2 expression in 50 SACCs and 20 normal salivary gland tissues nearby SACCs. The result showed that immunoreactivity for Nrp2 was detected in 47 of 50 SACCs, and its expression level had significant correlations with microvessel density, tumor size, TMN clinical stage, vascular invasion, and metastasis (P<0.05) of SACCs. In addition, inhibition of Nrp2 function by the receptor-ligand interaction-blocking antibody decreased cell migration, invasion, and angiogenic promotion without influences on the cell proliferation of Acc-3 cells. Taken together, the expression of Nrp2 protein is significantly correlated with tumor progression and angiogenesis in SACCs. These results suggest that Nrp2 may be a potential therapeutic target for SACCs., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Purpose: To investigate the potential inhibitory effects of RNA interference-mediated knockdown of neuropilin-2 (NP2) on inflammation-induced corneal hemangiogenesis and lymphangiogenesis, and whether selective inhibition of lymphangiogenesis on vascularized recipient beds before transplantation improves the graft survival., Methods: Mouse lymphatic endothelial cells were transfected with the plasmid expressing artificial microRNA (amiRNA) against mouse NP2, and the down-regulation of VEGF-C-induced NP2 expression by NP2 amiRNA was evaluated by real-time PCR and western blot assays. Next, NP2 amiRNA or negative control amiRNA was injected intrastromally into BALB/c mouse model of suture-induced corneal neovascularization three days after surgery. Corneas were harvested 1 week after suture placement and the formation of lymphatic and blood vessels as well as the recruitment of macrophage was evaluated by immunohistochemical staining. The neovascularized graft beds treated by NP2 amiRNA or control then served as recipients of orthotopic corneal transplants, and age-matched C57BL/6 donors were used. Corneal allografts were examined twice a week for 8 weeks, and graft clarity was quantified by means of an opacity score., Results: VEGF-C-induced NP2 expression at both mRNA and protein levels was significantly suppressed by NP2 amiRNA in mouse lymphatic endothelial cells. Intrastromal administration of NP2 amiRNA reduced corneal lymphangiogenesis by 45% versus control (p=0.015), but corneal hemangiogenesis (p=0.815) and the recruitment of CD11 antigen-like family member B (CD11b)-positive macrophage (p=0.589) were unchanged. Kaplan-Meier survival analysis revealed a better graft survival rate in the vascularized recipient beds pre-treated by NP2 amiRNA in comparison to controls (p=0.014)., Conclusions: Knockdown of NP2 improves corneal graft survival by selectively inhibiting lymphangiogenesis in vascularized beds before transplantation. Thus our results open new treatment options for transplant rejection and other lymphatic disorders.

Dendritic cell (DC) migration to secondary lymphoid organs is a critical step to properly exert its role in immunity and predominantly depends on the interaction of the chemokine receptor CCR7 with its ligands CCL21 and CCL19. Polysialic acid (PSA) has been recently reported to control CCL21-directed migration of mature DCs. Here, we first demonstrate that PSA present on human mature monocyte-derived dendritic cells did not enhance chemotactic responses to CCL19. We have also explored the molecular mechanisms underlying the selective enhancing effect of PSA on CCL21-driven chemotaxis of DCs. In this regard, we found out that prevention of DC polysialylation decreased CCL21 activation of JNK and Akt signaling pathways, both associated with CCR7-mediated chemotaxis. We also report that the enhanced PSA-mediated effect on DC migration towards CCL21 relied on the highly basic C-terminal region of this chemokine and depended on the PSA acceptor molecule neuropilin-2 (NRP2) and on the polysialyltransferase ST8SiaIV. Altogether, our data indicate that the CCR7/CCL21/NRP2/ST8SiaIV functional axis constitutes an important guidance clue for DC targeting to lymphoid organs.

Recent evidence shows that olfactory sensory neurons expressing a given odorant receptor (OR) are not necessarily confined to one of four zones, rather arranged in an overlapping manner in the olfactory epithelium (OE). In this study, in situ hybridization of OE sections with the OR probes indicated that the OR genes, the mRNAs of which were detected in an array of glomeruli on olfactory bulb (OB) along the anterodorsal/posteroventral (AD/PV) axis, are expressed in subareal zones within the most ventral zone, zone 4, along the dorsomedial/ventrolateral (DM/VL) axis. We also found that Neuropilin-2 (Nrp2) is expressed in a DM-low to VL-high gradient within zone 4 of OE. Furthermore, in Nrp2 mutant mice, we observed multiple glomeruli for zone 4 ORs in OB. These results suggest that the graded expression of Nrp2 in OE is required for the proper targeting of ventral glomeruli along the AD/PV axis in OB.

In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) roughly correlate with their axonal projection sites along the dorsal-ventral (D-V) axis of the olfactory bulb (OB). Here we report that an axon guidance receptor, Neuropilin-2 (Nrp2), and its repulsive ligand, Semaphorin-3F (Sema3F), are expressed by OSNs in a complementary manner that is important for establishing olfactory map topography. Sema3F is secreted by early-arriving axons of OSNs and is deposited at the anterodorsal OB to repel Nrp2-positive axons that arrive later. Sequential arrival of OSN axons as well as the graded and complementary expression of Nrp2 and Sema3F by OSNs help to form the topographic order along the D-V axis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Purpose: Compounds regulating intracellular thiol redox status, such as N,N-diacetyl-L-cystine dimethylester (NM(2)), were shown to prolong corneal graft survival in a penetrating keratoplasty (PKP) model. However, the effect of NM(2) on hemangiogenesis and lymphangiogenesis has not been investigated. The effect of manipulating ambient thiol redox status on riskier (higher rejection rate) transplantation models, such as limbal graft survival and hemangiogenesis and lymphangiogenesis in a corneal suture model, were investigated., Methods: C57BL/10 mice that received BALB/c corneas were treated by subconjunctival injection of NM(2), and limbal graft survival was assessed. Sutured C57BL/6 received daily intraperitoneal injections of NM(2), glutathione diethylester (GSH-OEt), or PBS. Lymphatic endothelial cell (LEC) and peritoneal mps were treated with NM(2) or GSHOEt, and then VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression were analyzed. Supernatants were collected for analysis of TNF-alpha and VEGF-A levels by ELISA., Results: Significantly less cellular infiltration was detected in mice with corneal limbal transplant-treated NM(2)-treated mice. Hemangiogenesis and lymphangiogenesis were suppressed in the NM(2)-treated mice. NM(2) treatment of mps led to reduced levels of VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression compared with PBS- or GSHOEt- treated mps, lower levels of TNF-alpha, and increased secretion of VEGF. Moreover, NM(2)-treated LECs had reduced levels of LYVE-1 and Prox-1., Conclusions: Reduction of ambient redox status reduced inflammatory cell infiltrates. Consequently, reduced inflammatory response might have contributed to both the observed prolonged corneal limbal graft survival and the attenuated hemangiogenesis and lymphangiogenesis in cornea.

Semaphorin 3F (Sema3F) is a secreted type of the semaphorin family of axon guidance molecules. Sema3F and its receptor Neuropilin-2 (Npn-2) mRNAs were distributed in a mutually exclusive manner throughout mouse brain development. In order to examine physiological roles of Sema3F, we generated Sema3F knockout mice (KO) by gene targeting in embryonic stem (ES) cells. We found that the loss of Sema3F expression did not significantly affect the mRNA expression of Npn-2 or the other putative Npn-2 ligands, namely, Sema3B, Sema3C, or Sema3G. The barrel structure of the somatosensory cortex and the cerebellar neuroanatomy were not significantly altered in Sema3F KO. Finally, optical imaging of intrinsic signals of the dorsal olfactory bulb showed no significant differences in odor map between wild-type mice and Sema3F KO. These data suggest that Sema3F plays a relatively restricted, if any, role in its receptor expression and postnatal development of these brain structures.

Background: After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2)., Results: We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents., Conclusions: RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.

Commissural axon guidance requires complex modulations of growth cone sensitivity to midline-derived cues, but underlying mechanisms in vertebrates remain largely unknown. By using combinations of ex vivo and in vivo approaches, we uncovered a molecular pathway controlling the gain of response to a midline repellent, Semaphorin3B (Sema3B). First, we provide evidence that Semaphorin3B/Plexin-A1 signaling participates in the guidance of commissural projections at the vertebrate ventral midline. Second, we show that, at the precrossing stage, commissural neurons synthesize the Neuropilin-2 and Plexin-A1 Semaphorin3B receptor subunits, but Plexin-A1 expression is prevented by a calpain1-mediated processing, resulting in silencing commissural responsiveness. Third, we report that, during floor plate (FP) in-growth, calpain1 activity is suppressed by local signals, allowing Plexin-A1 accumulation in the growth cone and sensitization to Sema3B. Finally, we show that the FP cue NrCAM mediates the switch of Plexin-A1 processing underlying growth cone sensitization to Sema3B. This reveals pathway-dependent modulation of guidance receptor processing as a novel mechanism for regulating guidance decisions at intermediate targets.

Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.

The majority of excitatory synapses in the mammalian CNS (central nervous system) are formed on dendritic spines, and spine morphology and distribution are critical for synaptic transmission, synaptic integration and plasticity. Here, we show that a secreted semaphorin, Sema3F, is a negative regulator of spine development and synaptic structure. Mice with null mutations in genes encoding Sema3F, and its holoreceptor components neuropilin-2 (Npn-2, also known as Nrp2) and plexin A3 (PlexA3, also known as Plxna3), exhibit increased dentate gyrus (DG) granule cell (GC) and cortical layer V pyramidal neuron spine number and size, and also aberrant spine distribution. Moreover, Sema3F promotes loss of spines and excitatory synapses in dissociated neurons in vitro, and in Npn-2(-/-) brain slices cortical layer V and DG GCs exhibit increased mEPSC (miniature excitatory postsynaptic current) frequency. In contrast, a distinct Sema3A-Npn-1/PlexA4 signalling cascade controls basal dendritic arborization in layer V cortical neurons, but does not influence spine morphogenesis or distribution. These disparate effects of secreted semaphorins are reflected in the restricted dendritic localization of Npn-2 to apical dendrites and of Npn-1 (also known as Nrp1) to all dendrites of cortical pyramidal neurons. Therefore, Sema3F signalling controls spine distribution along select dendritic processes, and distinct secreted semaphorin signalling events orchestrate CNS connectivity through the differential control of spine morphogenesis, synapse formation, and the elaboration of dendritic morphology.

Increased vascular permeability and vasodilation are responses usually elicited by snake envenomation. In this report, we isolated from Macroviperalebetina venom two protein groups designated IC1 (Increasing Capillary1) and IC2 based on their activities on capillary permeability. Mass spectrometry analysis showed that IC1 contained four major proteins of 23,650, 24,306, 24,589 and 24,718Da, whereas IC2 contained three major proteins of 25,101, 25,194 and 25,298Da. N-terminal amino-acid sequencing revealed that IC1 and IC2 belong to the snake venom VEGF (svVEGF) family. IC1 and IC2 had a marked specificity for VEGFR-2, with affinities in the nanomolar range. Interestingly, they also bind to NRP1 and NRP2, with affinities in the micromolar range. This is the first report demonstrating that M. lebetina encodes several distinct svVEGFs, endowed with a capacity to interact with neuropilins. IC1 and IC2 could be valuable tools to understand the molecular properties of angiogenic factors and their receptors.

Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are transmembrane glycoproteins with large extracellular domains that interact with class 3 semaphorins, vascular endothelial growth factor (VEGF) family members, and ligands, such as hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-beta1 (TGF-beta1), and fibroblast growth factor2 (FGF2). Neuropilins (NRPs) have been implicated in tumor growth and vascularization, as novel mediators of the primary immune response and in regeneration and repair; however, their role in renal pathophysiology is largely unknown. Here, we report upregulation of tubular and interstitial NRP2 protein expression in patients with focal segmental glomerulosclerosis (FSGS). In an additional cohort of patients with minimal change disease (MCD), membranous nephropathy (MN), and FSGS, elevated NRP2 mRNA expression in kidney biopsies inversely correlated with estimated glomerular filtration rate (eGFR) at the time of biopsy. Furthermore, upregulation of NRP2 mRNA correlated with post-bioptic decline of kidney function. Expression of NRP1 and NRP2 in human proximal tubular cells (PTCs) was differentially affected after stimulation with TGF-beta1, interleukin-1beta (IL-1beta), and oncostatin M (OSM). Although the pro-fibrotic mediators, TGF-beta1 and IL-1beta, induced upregulation of NRP2 expression but downregulation of NRP1 expression, OSM stimulated the expression of both NRP1 and NRP2. Basal and OSM-induced NRP1 mRNA expression, as well as TGF-beta1-induced NRP2 mRNA and protein expression were partially mediated by MEK1/2-ERK1/2 signaling. This is the first report suggesting a differential role of NRP1 and NRP2 in renal fibrogenesis, and TGF-beta1, IL-1beta, and OSM represent the first ligands known to stimulate NRP2 expression in mammalian cells.

Dopaminergic neurons in the mesodiencephalon (mdDA neurons) make precise synaptic connections with targets in the forebrain via the mesostriatal, mesolimbic, and mesoprefrontal pathways. Because of the functional importance of these remarkably complex ascending axon pathways and their implication in human disease, the mechanisms underlying the development of these connections are of considerable interest. Despite extensive in vitro studies, the molecular determinants that ensure the perfect formation of these pathways in vivo remain mostly unknown. Here, we determine the embryonic origin and ontogeny of the mouse mesoprefrontal pathway and use these data to reveal an unexpected requirement for semaphorin 3F (Sema3F) and its receptor neuropilin-2 (Npn-2) during mdDA pathway development using tissue culture approaches and analysis of sema3F(-/-), npn-2(-/-), and npn-2(-/-);TH-Cre mice. We show that Sema3F is a bifunctional guidance cue for mdDA axons, some of which have the remarkable ability to regulate their responsiveness to Sema3F as they develop. During early developmental stages, Sema3F chemorepulsion controls previously uncharacterized aspects of mdDA pathway development through both Npn-2-dependent (axon fasciculation and channeling) and Npn-2-independent (rostral growth) mechanisms. Later on, chemoattraction mediated by Sema3F and Npn-2 is required to orient mdDA axon projections in the cortical plate of the medial prefrontal cortex. This latter finding demonstrates that regulation of axon orientation in the target field occurs by chemoattractive mechanisms, and this is likely to also apply to other neural systems. In all, this study provides a framework for additional dissection of the molecular basis of mdDA pathway development and disease.

Aims: B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy that clinically ranges from indolent to rapidly progressive. CLL, like other cancers, can be affected by epigenetic alterations., Materials & Methods: A microarray discovery-based study was initiated to determine DNA methylation in CLL cases with a range of CD38 expression (1–92%)., Results: Many loci were either methylated or unmethylated across all CD38 levels, but differential methylation was also observed for some genes. Genomic sequencing of DLEU7 confirmed extensive cytosine methylation preferentially in patient samples with low CD38 expression, whereas NRP2, SFRP2 and ADAM12 were more commonly methylated in those with high CD38 expression., Conclusion: This study demonstrates that CLL is affected by CpG island methylation in some genes that segregate with CD38 expression levels, while most others show similar methylation patterns across all levels. The CpG island methylation in certain functional gene groups and pathway-associated genes that are known to be deregulated in CLL provides additional insights into the CLL methylome and epigenetic contribution to cellular dysfunction. It will now be useful to investigate the effectiveness of epigenetic therapeutic reversal of these alterations to develop effective treatments for the disease.

Objective: In inflammatory lesions, sympathetic nerve fibers disappear soon after the start of inflammation. We identified sympathetic nerve repellents as possible causal agents in rheumatoid arthritis (RA). On nerve terminals, repellent factors bind to neuropilin-2 and its coreceptor. The aim of this study was to investigate the role of neuropilin-2 in the synovial tissue of patients with RA and patients with osteoarthritis (OA) and in experimental arthritis., Methods: The density of neuropilin-2-positive fibers and cells positive for semaphorin 3F (a sympathetic repellent) was investigated using immunofluorescence staining. Enzyme-linked immunosorbent assay was used to detect soluble neuropilin-2 in body fluids from patients with RA and patients with OA. An axon outgrowth assay and a neuropilin-2 Fc fusion construct (neuropilin-2Fc) were used to investigate semaphorin 3F-induced sympathetic nerve repulsion. In an animal model of type II collagen-induced arthritis, soluble neuropilin-2Fc was studied in vivo., Results: The synovial density of neuropilin-2-positive sympathetic nerve fibers was lower in RA than in OA, but the density of cells positive for semaphorin 3F was similar. In synovial fluid, the level of soluble neuropilin-2 was markedly higher in RA compared with OA. Mouse sympathetic ganglia served as an excellent model with which to study semaphorin 3F-induced nerve fiber repulsion. Neuropilin-2 and its coreceptor were present on sympathetic neurons, and semaphorin 3F bound to neuropilin-2Fc (binding constant 96 nmoles/liter). Semaphorin 3F dose-dependently increased sympathetic nerve fiber repulsion (at a 50% maximum response concentration of 160-210 nmoles/liter). In contrast to our expectations, soluble neuropilin-2Fc did not inhibit repulsion but increased the repellent effect of semaphorin 3F. In experimental arthritis, therapy with neuropilin-2Fc aggravated arthritis., Conclusion: Soluble neuropilin-2 has no antirepellent activity but aggravates sympathetic nerve fiber repulsion and arthritis. Increased shedding of neuropilin-2 is probably an unfavorable sign in RA.

Recurrence and subsequent metastatic transformation of cancer develops from a subset of malignant cells, which show the ability to resist stress and to adopt to a changing microenvironment. These tumor cells have distinctly different growth factor pathways and antiapoptotic responses compared with the vast majority of cancer cells. Long-term therapeutic success can only be achieved by identifying and targeting factors and signaling cascades that help these cells survive during stress. Both microarray and immunohistochemical analysis on human prostate cancer tissue samples have shown an increased expression of vascular endothelial growth factor-C (VEGF-C) in metastatic prostate cancer. We have discovered that VEGF-C acts directly on prostate cancer cells to protect them against oxidative stress. VEGF-C increased the survival of prostate cancer cells during hydrogen peroxide stress by the activation of AKT-1/protein kinase Balpha. This activation was mediated by mammalian target of rapamycin complex-2 and was not observed in the absence of oxidative stress. Finally, the transmembrane nontyrosine kinase receptor neuropilin-2 was found to be essential for the VEGF-C-mediated AKT-1 activation. Indeed, our findings suggest a novel and distinct function of VEGF-C in protecting cancer cells from stress-induced cell death, thereby facilitating cancer recurrence and metastasis. This is distinctly different from the known function of VEGF-C in inducing lymphangiogenesis.

The structure and characteristics of the tumor vasculature are known to be different from those of normal vessels. Neuropilin2 (Nrp2), which is expressed in non-endothelial cell types, such as neuronal or cancer cells, functions as a receptor for both semaphorin and vascular endothelial growth factor (VEGF). After isolating tumor and normal endothelial cells from advanced gastric cancer tissue and normal gastric mucosa tissues, respectively, we identified genes that were differentially expressed in gastric tumor endothelial (TEC) and normal endothelial cells (NEC) using DNA oligomer chips. Using reverse transcriptase-PCR, we confirmed the chip results by showing that Nrp2 gene expression is significantly up-regulated in TEC. Genes that were found to be up-regulated in TEC were also observed to be up-regulated in human umbilical vein endothelial cells (HUVECs) that were co-cultured with gastric cancer cells. In addition, HUVECs co-cultured with gastric cancer cells showed an increased reactivity to VEGF-induced proliferation and migration. Moreover, overexpression of Nrp2 in HUVECs significantly enhanced the proliferation and migration induced by VEGF. Observation of an immunohistochemical analysis of various human tumor tissue arrays revealed that Nrp2 is highly expressed in the tumor vessel lining and to a lesser extent in normal tissue microvessels. From these results, we suggest that Nrp2 may function to increase the response to VEGF, which is more significant in TEC than in NEC given the differential expression, leading to gastric TEC with aggressive angiogenesis phenotypes.

Background: Malignant pleural mesothelioma is highly aggressive and recurs rapidly despite radical multimodality treatment. Progression of mesothelioma is thought to be governed by various growth factors, including vascular endothelial growth factor (VEGF). Placenta growth factor (PlGF) belongs to the VEGF family, although no study has yet investigated its expression in mesothelioma. We hypothesized that PlGF is overexpressed in mesothelioma and could have prognostic value in patients treated by extrapleural pneumonectomy., Methods: We assessed by immunohistochemistry with semiquantitative classification (0 = no staining; 3 = strong staining), the expression levels of PlGF and its cognate receptors VEGF receptor 1, neuropilin-1, and neuropilin-2 in 27 patients with malignant pleural mesothelioma undergoing extrapleural pneumonectomy, in 14 patients with reactive mesothelium, and in 10 patients with normal mesothelium., Results: Whereas PlGF was not expressed in normal mesothelium, it was overexpressed (grade 3) more frequently in mesothelioma than in reactive mesothelium specimens (11 or 41% versus 1 or 7%, respectively, p = 0.03). Furthermore, in mesothelioma, VEGF receptor 1 and neuropilin-1 and -2 were overexpressed in 18 specimens (67%), 8 specimens (30%), and 9 specimens (33%), respectively. Mean survival after extrapleural pneumonectomy was 17 months. An inverse relationship was found between the degree of PlGF expression and survival in months (R = -0.45, p = 0.01). No correlation was found between tumor stage and survival (R = -0.33) and between tumor stage and PlGF expression (R = 0.07)., Conclusions: We have shown that PlGF can be overexpressed in malignant pleural mesothelioma. In addition, the finding of an inverse relationship between PlGF expression levels and survival suggests a pivotal role of this factor in the recurrence and progression of mesothelioma after extrapleural pneumonectomy.

Intra-amniotic infection/inflammation (IAI) is a major cause of preterm birth, but the mechanisms responsible are not well understood. This study investigates the effects of IAI on vascular endothelial growth factor (VEGF) as well as VEGF receptor (Flt1, KDR2) and coreceptor (neuropilin-1 and -2) messenger RNA (mRNA) and protein expression at the maternal-fetal interface, both in vitro and in vivo. Decidual stromal cells (DSCs) were isolated from term placentae, purified, and treated with 10(-8) mol/L estradiol (E(2)), 10( -7) mol/L medroxyprogesterone acetate (MPA), both, or vehicle for 7 days. Vascular endothelial growth factor expression in cultured DSCs increased in response to stimulation with interleukin 1 beta (IL-1 beta; 0.01-10 ng/mL)--but not tumor necrosis factor alpha (TNF-alpha; 1 ng/mL)--in a concentration-dependent fashion irrespective of the hormonal milieu. This effect appears to be mediated at the level of gene transcription because stimulation with IL-1 beta (but not TNF-alpha) increased expression of VEGF mRNA as measured by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR); a similar increase was seen in neuropilin-1/-2 (but not Flt1 and KDR2) mRNA. Immunohistochemical studies confirmed these observations in vivo. Immunostaining for VEGF and neuropilin-1/-2 (but not Flt1 or KDR2) was increased in serial tissue sections of decidua from women with clinical and histological evidence of IAI versus noninfected controls, and in cultured term DSCs exposed to IL-1 beta. The novel observations that IL-1 beta stimulates VEGF and neuropilin-1/-2 mRNA and protein expression in term DSCs in vitro along with confirmatory in vivo data using immunohistochemistry provide a mechanism by which IAI can alter vascular permeability, thereby facilitating leukocyte trafficking and increasing the risk of abruption, both of which are associated with preterm birth.

Background: Profound hearing loss is a serious complication of neurofibromatosis type 2, a genetic condition associated with bilateral vestibular schwannomas, benign tumors that arise from the eighth cranial nerve. There is no medical treatment for such tumors., Methods: We determined the expression pattern of vascular endothelial growth factor (VEGF) and three of its receptors, VEGFR-2, neuropilin-1, and neuropilin-2, in paraffin-embedded samples from 21 vestibular schwannomas associated with neurofibromatosis type 2 and from 22 sporadic schwannomas. Ten consecutive patients with neurofibromatosis type 2 and progressive vestibular schwannomas who were not candidates for standard treatment were treated with bevacizumab, an anti-VEGF monoclonal antibody. An imaging response was defined as a decrease of at least 20% in tumor volume, as compared with baseline. A hearing response was defined as a significant increase in the word-recognition score, as compared with baseline., Results: VEGF was expressed in 100% of vestibular schwannomas and VEGFR-2 in 32% of tumor vessels on immunohistochemical analysis. Before treatment, the median annual volumetric growth rate for 10 index tumors was 62%. After bevacizumab treatment in the 10 patients, tumors shrank in 9 patients, and 6 patients had an imaging response, which was maintained in 4 patients during 11 to 16 months of follow-up. The median best response to treatment was a volumetric reduction of 26%. Three patients were not eligible for a hearing response; of the remaining seven patients, four had a hearing response, two had stable hearing, and one had progressive hearing loss. There were 21 adverse events of grade 1 or 2., Conclusions: VEGF blockade with bevacizumab improved hearing in some, but not all, patients with neurofibromatosis type 2 and was associated with a reduction in the volume of most growing vestibular schwannomas., (2009 Massachusetts Medical Society)

The peripheral nervous system (PNS) of higher vertebrates is segmented to align the spinal nerve roots with the vertebrae. This co-patterning is set up during embryogenesis, when vertebrae develop from the sclerotome layer of the metameric somites, and PNS neurons and glia differentiate from neural crest cells (NCCs) that preferentially migrate into the anterior sclerotome halves. Previous analyses of mice deficient in the class 3 semaphorin (SEMA3) receptors neuropilin (NRP) 1 or 2 raised the possibility that each controlled a distinct aspect of trunk NCC migration. We now demonstrate that both pathways act sequentially in distinct NCC subpopulations and thereby cooperate to enforce segmental NCC migration. Specifically, SEMA3A/NRP1 signalling first directs one population of NCCs from the intersomitic path into the sclerotome, and SEMA3F/NRP2 signalling acts subsequently to restrict a second population to the anterior half of the sclerotome. NCC exclusion from either the posterior sclerotome or the intersomitic boundary is sufficient to enforce the separation of neighbouring NCC streams and the segregation of sensory NCC progeny into metameric dorsal root ganglia (DRG). By contrast, the combined loss of both guidance pathways leads to ectopic invasion of the intersomitic furrows and posterior sclerotome halves, disrupting metameric NCC streaming and DRG segmentation.

Semaphorins are a large family of secreted and membrane-bound proteins. Recently, several roles of semaphorins in the immune system have emerged. Several semaphorins and their receptors are expressed in a variety of lymphoid and myeloid cells and affect immune cell functions, including cell proliferation, differentiation, chemotaxis, and cytokine production. However, the roles of class 3 semaphorins in human myeloid cells are not well known. Here we examined the regulation of expression of class 3 semaphorins and their receptors by inflammatory stimuli and their function in human macrophages. We show that the expression of Sema3A receptors (neuropilin-1 (NRP-1), NRP-2, plexin A1, plexin A2, and plexin A3) significantly increased during M-CSF-mediated differentiation of monocytes into macrophages under conditions that promote an M2 alternatively activated macrophage phenotype. Consistent with increased NRP-1 expression, cell surface binding of Sema3A increased during M2 differentiation. Interferon (IFN)-gamma and lipopolysaccharide, which promote classical M1 macrophage activation affected expression of NRP-1, NRP-2 and plexin A1. IFN-gamma decreased NRP-1 expression and LPS suppressed NRP-2 and plexin A1 expression. Furthermore we show that Sema3A induced apoptosis in monocyte-derived macrophages and cooperated with anti-Fas CH11 antibody to augment apoptosis. Our results suggest that Sema3A plays a role in induction of apoptosis in monocyte-derived macrophages that are resistant to Fas-induced apoptosis, and that its function can be modulated in inflammatory conditions.

Placenta growth factor (PlGF) is a key regulator of pathological angiogenesis and its overexpression has been linked to neoplastic progression. To assess whether PlGF could have a role in malignant mesothelioma (MM), we analyzed the expression of PlGF, VEGF, and their cognate receptors (VEGF-R1 and VEGF-R2) and co-receptors (neuropilin-1 and neuropilin-2) in MM cell lines as well as in resected MM tissues, hyperplastic/reactive mesothelium and normal mesothelium. MM cell cultures expressed both ligands and the associated receptors to a variable extent and released different amounts of PlGF. As assessed by immunohistochemistry, PlGF expression was switched on in hyperplastic/reactive compared to normal mesothelium. Moreover, 74 and 94 percent of MM tissues overexpressed PlGF and VEGF-R1, respectively (p<0.05 MM vs normal mesothelium). Administration of recombinant PlGF-2 did not elicit a significant stimulation of MM cell growth, while it was associated with a transient phosphorylation of Akt, suggesting that PlGF-2 could activate downstream effectors of proliferative and cytoprotective signals via VEGF-R1 in MM cells. Indeed, the administration of an anti-PlGF antibody was found to cause a significant reduction of MM cell survival. In conclusion, our data demonstrate that, by acting as a survival factor, PlGF can play a role which goes beyond the stimulation of angiogenesis in MM. This evidence could help the rational design of new therapeutic interventions for this aggressive tumor.

Accumulating evidence indicates that signaling centers controlling the dorsoventral (DV) polarization of the neural tube, the roof plate and the floor plate, play crucial roles in axon guidance along the DV axis. However, the role of signaling centers regulating the rostrocaudal (RC) polarization of the neural tube in axon guidance along the RC axis remains unknown. Here, we show that a signaling center located at the midbrain-hindbrain boundary (MHB) regulates the rostrally directed growth of axons from midbrain dopaminergic neurons (mDANs). We found that beads soaked with fibroblast growth factor 8 (FGF8), a signaling molecule that mediates patterning activities of the MHB, repelled mDAN axons that extended through the diencephalon. This repulsion may be mediated by semaphorin 3F (sema3F) because (1) FGF8-soaked beads induced an increase in expression of sema3F, (2) sema3F expression in the midbrain was essentially abolished by the application of an FGF receptor tyrosine kinase inhibitor, and (3) mDAN axonal growth was also inhibited by sema3F. Furthermore, mDAN axons expressed a sema3F receptor, neuropilin-2 (nrp2), and the removal of nrp-2 by gene targeting caused caudal growth of mDAN axons. These results indicate that the MHB signaling center regulates the growth polarity of mDAN axons along the RC axis by inducing sema3F.

Background: Multicellular resistance (MCR), i.e. decreased sensitivity to anticancer drugs compared with common monolayer cell (MC) cultures, depends partly on tumor cell-cell adhesion. Previous studies have shown that anti-adhesive therapies, including integrin alpha(v), beta(1) and alpha(v)beta(3) targeting, induced apoptosis and reversed the sensitivity of MCR., Methods: A model of three-dimensional cell culture was used to establish HT29 multicellular spheroid cells (MCS) and explore the effect of semaphorin3F (Sema3F) on integrin-mediated cell-cell interactions in MCS of a human colorectal adenocarcinoma cell line (HT29) and sensitization of HT29 MCS to 5-fluorouracil and oxaliplatin via a decrease in integrin alpha(v)beta(3)., Results: Elevated expression of Sema3F led to the up-regulation neuropilin-2 (Nrp2) receptor expression and the down-regulation of integrin alpha(v)beta(3) expression. Furthermore, short interfering RNA of Nrp2 could reverse MCR., Conclusion: Our study demonstrates that Sema3F can sensitize MCR by decreasing integrin alpha(v)beta(3)expression via the Nrp2 receptor., (Copyright 2009 S. Karger AG, Basel.)

Neural representations of the environment within the brain take the form of topographic maps whose formation relies on graded expression of axon guidance molecules. Retinocollicular map formation, from retinal ganglion cells (RGCs) to the superior colliculus (SC) in the midbrain, is mainly driven by Eph receptors and their ligands ephrins. However, other guidance molecules participate in the formation of this map. Here we demonstrate that the receptor Neuropilin-2 is expressed in an increasing nasal-temporal gradient in RGCs, whereas one of its ligands, Semaphorin3F, but not other Sema3 molecules, presents a graded low-rostral to high-caudal expression in the SC when mapping is underway. Neuropilin-2 and its coreceptor Plexin A1 are present on RGC growth cones. Collapse assays demonstrate that Semaphorin3F induces significant growth cone collapse of temporal, but not nasal, RGCs expressing high levels of Neuropilin-2. Our results suggest that Neuropilin-2/Semaphorin3F are new candidates involved in retinotopy formation within the SC., (Copyright (c) 2008 Wiley-Liss, Inc.)

Peripheral nerve transection or crush induces expression of class 3 semaphorins by epineurial and perineurial cells at the injury site and of the neuropilins neuropilin-1 and neuropilin-2 by Schwann and perineurial cells in the nerve segment distal to the injury. Neuropilin-dependent class 3 semaphorin signaling guides axons during neural development, but the significance of this signaling system for regeneration of adult peripheral nerves is not known. To test the hypothesis that neuropilin-2 facilitates peripheral-nerve axonal regeneration, we crushed sciatic nerves of adult neuropilin-2-deficient and littermate control mice. Axonal regeneration through the crush site and into the distal nerve segment, repression by the regenerating axons of Schwann cell p75 neurotrophin receptor expression, remyelination of the regenerating axons, and recovery of normal gait were all significantly slower in the neuropilin-2-deficient mice than in the control mice. Thus, neuropilin-2 facilitates peripheral-nerve axonal regeneration., ((c) 2008 Wiley-Liss, Inc.)

The neuropilins-1 and -2 (NRP1 and NRP2) function as receptors for both the semaphorins and vascular endothelial growth factor. In addition to their contribution to the development of the nervous system, NRP1 and NRP2 have been implicated in angiogenesis and tumor progression. Given their importance to cancer and endothelial biology and their potential as therapeutic targets, an important issue that has not been addressed is the impact of metabolic stress conditions characteristic of the tumor microenvironment on their expression and function. Here, we demonstrate that hypoxia and nutrient deprivation stimulate the rapid loss of NRP1 expression in both endothelial and carcinoma cells. NRP2 expression, in contrast, is maintained under these conditions. The lysosomal inhibitors chloroquine and bafilomycin A1 prevented the loss of NRP1 expression, but proteasomal inhibitors had no effect. The hypothesis that NRP1 is degraded by autophagy is supported by the findings that its expression is lost rapidly in response to metabolic stress, prevented with 3-methyladenine and induced by rapamycin. Targeted depletion of NRP2 using small hairpin RNA revealed that NRP2 can function in the absence of NRP1 to mediate endothelial tube formation in hypoxia. Studies aimed at assessing NRP function and targeted therapy in cancer and angiogenesis should consider the impact of metabolic stress.

Class three semaphorins (SEMAs) were originally shown to be mediators of axon guidance that repelled axons and collapsed growth cones, but it is now evident that SEMA3F, for example, has similar effects on tumor cells and endothelial cells (EC). In both human U87MG glioma cells and human umbilical vein EC, SEMA3F induced rapid cytoskeletal collapse, suppressed cell contractility, decreased phosphorylation of cofilin, and inhibited cell migration in culture. Analysis of the signaling pathways showed that SEMA3F formed a complex with NRP2 (neuropilin-2) and plexin A1. These interactions eventually led to inactivation of the small GTPase, RhoA, which is necessary for stress fiber formation and cytoskeleton integrity. A novel upstream RhoA mediator was shown to be ABL2, also known as ARG, a membrane-anchored nonreceptor tyrosine kinase. Within minutes after the addition of SEMA3F, ABL2 directly bound plexin A1 but not to a plexin A1 mutant lacking the cytoplasmic domain. In addition, ABL2 phosphorylated and thereby activated p190RhoGAP, which inactivated RhoA (GTP to GDP), resulting in cytoskeleton collapse and inhibition of cell migration. On the other hand, cells overexpressing an ABL2 inactive kinase mutant or treated with ABL2 small interfering RNA did not inactivate RhoA. Cells treated with p190RhoGAP small interfering RNA also did not inactivate RhoA. Together, these results suggested that ABL2/ARG is a novel mediator of SEMA3F-induced RhoA inactivation and collapsing activity.

VEGF-C is a crucial player in lymphangiogenesis. Besides VEGFR-2 and VEGFR-3, it also binds NRP2. NRP2 enhances VEGF-C/VEGFR-3 effects in developmental lymphangiogenesis, but its role in adult and tumoral lymphangiogenesis is not known. In their study, Bagri and colleagues demonstrate that blocking NRP2 results in a decrease of metastasis formation, a phenomenon relying on tumoral lymphangiogenesis. Thus, they identified NRP2 as an attractive new target for modulating metastasis.

The homeodomain transcription factor Nkx2-1 plays key roles in the developing telencephalon, where it regulates the identity of progenitor cells in the medial ganglionic eminence (MGE) and mediates the specification of several classes of GABAergic and cholinergic neurons. Here, we have investigated the postmitotic function of Nkx2-1 in the migration of interneurons originating in the MGE. Experimental manipulations and mouse genetics show that downregulation of Nkx2-1 expression in postmitotic cells is necessary for the migration of interneurons to the cortex, whereas maintenance of Nkx2-1 expression is required for interneuron migration to the striatum. Nkx2-1 exerts this role in the migration of MGE-derived interneurons by directly regulating the expression of a guidance receptor, Neuropilin-2, which enables interneurons to invade the developing striatum. Our results demonstrate a role for the cell-fate determinant Nkx2-1 in regulating neuronal migration by direct transcriptional regulation of guidance receptors in postmitotic cells.

Background: The development of the corticospinal tract (CST) in higher vertebrates relies on a series of axon guidance decisions along its long projection pathway. Several guidance molecules are known to be involved at various decision points to regulate the projection of CST axons. However, previous analyses of the CST guidance defects in mutant mice lacking these molecules have suggested that there are other molecules involved in CST axon guidance that are yet to be identified. In this study, we investigate the role of plexin signaling in the guidance of motor CST axons in vivo., Results: Expression pattern studies show that plexin-A3, plexin-A4, and neuropilin-1 are expressed in the developing cerebral cortex when the motor CST axons originating from layer V cortical neurons are guided down to the spinal cord. By analyzing mutant mice, we show that motor CST axons that turn dorsally to cross the midline at the pyramidal decussation require plexin-A3 and plexin-A4 signaling. Although other CST guidance defects are found in neuropilin-1 mutants, this dorsal turning defect is not observed in either neuropilin-1 or neuropilin-2 mutants, suggesting that the local cues that activate plexin signaling at the dorsal turning point are membrane-bound semaphorins. Further expression pattern study and mutant analysis indicate that Sema6A is one of the local cues for motor CST axon turning at the pyramidal decussation., Conclusion: Dorsal turning and midline crossing at the pyramidal decussation is a crucial step to properly direct CST axons into the dorsal spinal cord. We show that the signaling of plexin-A3, plexin-A4, and Sema6A is at least partially required for dorsal turning of the CST axons, while neuropilin-1 is required for proper fasciculation of the tract at midline crossing. Together with previous reports, these results demonstrate that several guidance cues are specifically utilized to regulate the dorsal turning and midline crossing of developing CST axons.

Neurons in the developing CNS tend to send out long axon collaterals to multiple target areas. For these neurons to attain specific connections, some of their axon collaterals are subsequently pruned-a process called stereotyped axon pruning. One of the most striking examples of stereotyped pruning in the CNS is the pruning of corticospinal tract (CST) axons. The long CST collaterals from layer V neurons of the visual and motor cortices are differentially pruned during development. Here we demonstrate that select plexins and neuropilins, which serve as coreceptors for semaphorins, are expressed in visual cortical neurons at the time when CST axon collaterals are stereotypically pruned. By analyzing mutant mice, we find that the pruning of visual, but not motor, CST axon collaterals depends on plexin-A3, plexin-A4, and neuropilin-2. Expression pattern study suggests that Sema3F is a candidate local cue for the pruning of visual CST axons. Using electron microscopic analysis, we also show that visual CST axon collaterals form synaptic contacts in the spinal cord before pruning and that the unpruned collaterals in adult mutant mice are unmyelinated and maintain their synaptic contacts. Our results indicate that the stereotyped pruning of the visual and motor CST axon collaterals is differentially regulated and that this specificity arises from the differential expression of plexin receptors in the cortex.

Interleukin-8 (IL-8) is associated with tumorigenesis by promoting angiogenesis and metastasis. Although up-regulation of IL-8 is indicated in many cancers, its function in pancreatic cancer has not been well characterized. In this study we examined the expression of IL-8 on pancreatic cancer cells and clinical tissue specimens, and investigated the effect of exogenous IL-8 on gene expression, and signaling in human pancreatic cancer cells. We found that pancreatic cancer cells expressed higher amount of IL-8 mRNA than normal human pancreatic ductal epithelium cells. IL-8 mRNA was also substantially overexpressed in 11 of 14 (79%) clinical pancreatic-adenocarcinoma samples compared with that in their surrounding normal tissues. Exogenous IL-8 up-regulated the expression of vascular endothelial growth factor(165), and neuropilin (NRP)-2 in BxPC-3 cells, one of human pancreatic cancer cell lines. IL-8 expression was inducible by hypoxia mimicking reagent cobalt chloride. In addition, IL-8 activated extracellular signal-regulated kinase (ERK)1/2 signaling pathway in BxPC-3 cells. Our studies suggest that IL-8 might be a malignant factor in human pancreatic cancer by induction of vascular endothelial growth factor and NRP-2 expression and ERK activation. Targeting IL-8 along with other antiangiogenesis therapy could be an effective treatment for this malignancy.

Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.

Neuropilin-1 and -2 (NRP1 and NRP2) are the transmembrane glycoproteins interacting with 2 types of ligands: class III semaphorins and several members of the VEGF family, the main regulators of blood and lymphatic vessel growth. We show here that both NRP1 and NRP2 can also bind hepatocyte growth factor (HGF). HGF is a pleiotropic cytokine and potent proangiogenic molecule that acts on its target cells by binding to the c-met receptor. We found that the N-terminal domain of HGF is involved in the interaction with neuropilins. We demonstrated that invalidation of NRP1 or NRP2 by RNA interference in human umbilical vein endothelial cells (HUVECs) decreased HGF-induced c-met phosphorylation and VEGF-A(165)- and HGF-mediated intracellular signaling. Accordingly, the disruption of NRP1 or NRP2 binding to VEGF-A(165) or HGF with a blocking antibody, decreased the proliferation and migration of endothelial cells. This effect may be further enhanced if VEGF-A(165) or HGF binding to both NRP1 and NRP2 was disrupted. Using a mouse Matrigel model, we demonstrated that NRP1 is essential for HGF-mediated angiogenesis in vivo. Our results suggest that, in endothelial cells, both NRP1 and NRP2 function as proangiogenic coreceptors, potentiating the activity of at least 2 major proangiogenic cytokines, VEGF-A(165) and HGF.

By analyzing the mechanisms that govern dopaminergic axon pathfinding from the midbrain to the striatum in embryonic rat brains, we identified neuroepithelial regions that exert chemotropic effects on mesencephalic dopaminergic axons. Explants from the pretectum and the striatum showed an attractive effect, whereas those from the midhindbrain boundary, the dorsal thalamus, and the ventral thalamus had no effect. Expression of semaphorin (Sema) 3C and Sema3F in the pretectum and of Sema3A in the striatum suggested a role for these axon guidance molecules in dopaminergic axon pathfinding. When expressed in HEK293 cell aggregates, Sema3C had an attractive effect and enhanced axon growth, Sema3A enhanced axon growth, and Sema3F had a repulsive effect on dopaminergic axons. Antineuropilin-1 and antineuropilin-2 antibodies reduced attraction by the pretectum, whereas attraction by the striatum was not affected by the presence of antineuropilin-1 antibodies. Moreover, neuropilin-1- and neuropilin-2-soluble Fc chimeras reduced the attraction by the pretectum. These results suggest that semaphorins may help to establish the dopaminergic projection from the midbrain to the striatum during embryonic development.

Background: Neuropilin-2 (NRP2) is a high-affinity kinase-deficient receptor for vascular endothelial growth factor (VEGF) and semaphorin 3F. We investigated its function in human colorectal cancers., Methods: Immunohistochemistry and immunoblotting were used to assess NRP2 expression levels in colorectal tumors and colorectal cancer cell lines, respectively. HCT-116 colorectal cancer cells stably transfected with short hairpin RNA (shRNAs) against NRP2 or control shRNAs were assayed for proliferation by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and for activation of the VEGFR1 pathway by immunoblotting. Soft agar assays, Annexin V staining, and Boyden chamber assays were used to examine anchorage-independent growth, apoptosis in response to hypoxia, and cell migration/invasion, respectively, in HCT-116 transfectants. Tumor growth and metastasis were analyzed in mice (groups of 10) injected with shRNA-expressing HCT-116 cells. The effect of in vivo targeting of NRP2 by small interfering RNA (siRNA) on the growth of hepatic colorectal tumors derived from luciferase-expressing HCT-116 cells was assessed by measuring changes in bioluminescence and final tumor volumes. All statistical tests were two-sided., Results: NRP2 expression was substantially higher in tumors than in adjacent mucosa. HCT-116 transfectants with reduced NRP2 levels had reduced VEGFR1 signaling, but proliferation was unchanged. Anchorage-independent growth, survival under hypoxic conditions, and motility/invasiveness were also reduced. In vivo, HCT-116 transfectants with reduced NRP2 demonstrated decreased tumor growth, fewer metastases, and increased apoptosis compared with control cells. Hepatic colorectal tumors in mice treated with NRP2 siRNAs were statistically significantly smaller than those in mice treated with control siRNAs (at 28 days after implantation, mean control siRNAs = 420 mm3, mean NRP2 siRNAs = 36 mm3, NRP2 vs control: difference = 385 mm3, 95% confidence interval = 174 mm3 to 595 mm3, P = .005)., Conclusion: NRP2 on colorectal carcinoma cells is important for tumor growth and is a potential therapeutic target in human cancers where it is expressed.

Background: In developing neurons, somal migration and initiation of axon outgrowth often occur simultaneously and are regulated in part by similar classes of molecules. When neurons reach their final destinations, however, somal translocation and axon extension are uncoupled. Insights into the mechanisms underlying this process of disengagement came from our study of the behaviour of embryonic spinal motor neurons following ablation of boundary cap cells. These are neural crest derivatives that transiently reside at motor exit points, central nervous system (CNS):peripheral nervous system (PNS) interfaces where motor axons leave the CNS. In the absence of boundary cap cells, motor neuron cell bodies migrate along their axons into the periphery, suggesting that repellent signals from boundary cap cells regulate the selective gating of somal migration and axon outgrowth at the motor exit point. Here we used RNA interference in the chick embryo together with analysis of null mutant mice to identify possible boundary cap cell ligands, their receptors on motor neurons and cytoplasmic signalling molecules that control this process., Results: We demonstrate that targeted knock down in motor neurons of Neuropilin-2 (Npn-2), a high affinity receptor for class 3 semaphorins, causes their somata to migrate to ectopic positions in ventral nerve roots. This finding was corroborated in Npn-2 null mice, in which we identified motor neuron cell bodies in ectopic positions in the PNS. Our RNA interference studies further revealed a role for Plexin-A2, but not Plexin-A1 or Plexin-A4. We show that chick and mouse boundary cap cells express Sema3B and 3G, secreted semaphorins, and Sema6A, a transmembrane semaphorin. However, no increased numbers of ectopic motor neurons are found in Sema3B null mouse embryos. In contrast, Sema6A null mice display an ectopic motor neuron phenotype. Finally, knockdown of MICAL3, a downstream semaphorin/Plexin-A signalling molecule, in chick motor neurons led to their ectopic positioning in the PNS., Conclusion: We conclude that semaphorin-mediated repellent interactions between boundary cap cells and immature spinal motor neurons regulates somal positioning by countering the drag exerted on motor neuron cell bodies by their axons as they emerge from the CNS at motor exit points. Our data support a model in which BC cell semaphorins signal through Npn-2 and/or Plexin-A2 receptors on motor neurons via a cytoplasmic effector, MICAL3, to trigger cytoskeletal reorganisation. This leads to the disengagement of somal migration from axon extension and the confinement of motor neuron cell bodies to the spinal cord.

Vascular endothelial growth factor C (VEGF-C) is a lymphangiogenic factor over-expressed in highly metastatic, cyclooxygenase (COX)-2 expressing breast cancer cells. We tested the hypothesis that tumour-derived VEGF-C may play an autocrine role in metastasis by promoting cellular motility through one or more VEGF-C-binding receptors VEGFR-2, VEGFR-3, neuropilin (NRP)-1, NRP-2, and integrin alpha9beta1. We investigated the expression of these receptors in several breast cancer cell lines (MDA-MB-231, Hs578T, SK-BR-3, T-47D, and MCF7) and their possible requirement in migration of two VEGF-C-secreting, highly metastatic lines MDA-MB-231 and Hs578T. While cell lines varied significantly in their expression of above VEGF-C receptors, migratory activity of MDA-MB-231 and Hs578T cells was linked to one or more of these receptors. Depletion of endogenous VEGF-C by treatments with a neutralising antibody, VEGF-C siRNA or inhibitors of Src, EGFR/Her2/neu and p38 MAP kinases which inhibited VEGF-C production, inhibited cellular migration, indicating the requirement of VEGF-C for migratory function. Migration was differentially attenuated by blocking or downregulation of different VEGF-C receptors, for example treatment with a VEGFR-2 tyrosine kinase inhibitor, NRP-1 and NRP-2 siRNA or alpha9beta1 integrin antibody, indicating the participation of one or more of the receptors in cell motility. This novel role of tumour-derived VEGF-C indicates that breast cancer metastasis can be promoted by coordinated stimulation of lymphangiogenesis and enhanced migratory activity of breast cancer cells.

Polysialic acid (PSA) is a unique linear homopolymer of alpha2,8-linked sialic acid that has been identified as a posttranslational modification on only five mammalian proteins. Studied predominantly on neural cell adhesion molecule (NCAM) during development of the vertebrate nervous system, PSA modulates cell interactions mediated by NCAM and other adhesion molecules. An isoform of NCAM (CD56) on natural killer (NK) cells is the only protein known to be polysialylated in cells of the immune system, yet the function of PSA in NK cells remains unclear. We show here that neuropilin-2 (NRP-2), a receptor for the semaphorin and vascular endothelial growth factor families in neurons and endothelial cells, respectively, is expressed on the surface of human dendritic cells and is polysialylated. Expression of NRP-2 is up-regulated during dendritic cell maturation, coincident with increased expression of ST8Sia IV, one of the key enzymes of PSA biosynthesis, and with the appearance of PSA on the cell surface. PSA on NRP-2 is resistant to digestion with peptide N-glycosidase F but is sensitive to release under alkaline conditions, suggesting that PSA chains are added to O-linked glycans of NRP-2. Removal of polysialic acid from the surface of dendritic cells or binding of NRP-2 with specific IgG promoted dendritic cell-induced activation and proliferation of T lymphocytes. Thus, this newly recognized polysialylated protein on the surface of dendritic cells influences dendritic cell-T lymphocyte interactions through one or more of its distinct extracellular domains.

Semaphorin-3A (sema3A) is a neuropilin-1 (np1) agonist. It inhibits the binding of the 165-amino acid form of VEGF (VEGF(165)) to np1 and was reported to inhibit angiogenesis as a result. However, we find that sema3A concentrations that inhibit the mitogenic effects of VEGF(165) do not inhibit VEGF(165)-induced phosphorylation of VEGF receptor-2 (VEGFR-2). Furthermore, sema3A inhibits the biological effects of VEGF(121), a VEGF form that does not bind to neuropilins and basic fibroblast growth factor, a growth factor whose activity, unlike that of VEGF, is not inhibited by small interfering RNA directed against np1. Therefore, the mechanism by which sema3A inhibits VEGF(165) activity does not depend on competition with VEGF(165) for binding to np1. Sema3A induced rapid disappearance of focal contacts followed by collapse of the actin cytoskeleton in human umbilical vein-derived endothelial cells. HEK293 cells expressing sema3A repel human endothelial cells and at high concentrations induce their death by apoptosis. Furthermore, sema3A inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay. Similar effects are induced by the neuropilin-2 (np2) agonist sema3F. These inhibitory effects are abrogated by small interfering RNAs directed against np1 or np2, respectively. The anti-proliferative effects of sema3A and sema3F are additive when the semaphorins are added as pure proteins. However, when sema3A and sema3F were co-expressed in HEK293 cells their pro-apoptotic and cell repellant activities appeared to be synergistic. These observations suggest that combinations of sema3A and sema3F may be able to inhibit tumor angiogenesis more effectively than single semaphorins.

Neuropilins (NRPs) are 130-kDa receptors that bind and respond to the class 3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF165 and SEMA3F. Cell binding studies reveal that VEGF165 and SEMA3F bind NRP2 with similar affinities, 5.2 and 3.9 nM, respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF165 binding, whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF165 binding, point mutations were introduced by site-directed mutagenesis. VEGF165 is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A,E291A) in the 280-290 b1-NRP2 loop resulted in a 2-fold reduction in VEGF165 affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E,N290D and R287E,N290S) significantly increased VEGF165 affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF165 affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF165 binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF165/NRP2 interactions.

The neuropilin-1 (np1) receptor binds the 165 amino-acid form of vascular endothelial growth factor165 (VEGF165) and functions as an enhancer that potentiates VEGF165 signaling via the VEGFR-2 tyrosine-kinase receptor. To study the mechanism by which neuropilins potentiate VEGF activity we produced a VEGF165 mutant (VEGF165KF) that binds to neuropilins but displays a much lower affinity toward VEGFR-1 and VEGFR-2. VEGF165KF failed to induce VEGFR-2 phosphorylation in cells lacking neuropilins. However, in the presence of np1, VEGF165KF bound weakly to VEGFR-2, induced VEGFR-2 phosphorylation, and activated ERK1/2. Interestingly, VEGF165KF did not promote formation of VEGFR-2/np1 complexes nor did high concentrations of VEGF165KF inhibit VEGF165 induced formation of such complexes, suggesting that VEGF165 does not stabilize VEGFR-2/np1 complexes by forming bridges spanning VEGFR-2 and np1. VEGF121 is a VEGF form that does not bind to neuropilins. Surprisingly, both np1 and neuropilin-2 (np2) enhanced VEGF121-induced phosphorylation of VEGFR-2 and VEGF121-induced proliferation of endothelial cells. The enhancement of VEGF121 activity by np1 was accompanied by a 10-fold increase in binding affinity to VEGFR-2 and was not associated with the formation of new VEGFR-2/np1 complexes. These observations suggest that neuropilins enhance the activity of VEGF forms that do not bind to neuropilins, indicate that np2 is a functional VEGF receptor, and imply that spontaneously formed VEGFR-2/np1 complexes suffice for efficient neuropilin mediated enhancement of VEGF activity.

Although the regulation of tumor angiogenesis is believed to be one of the core functions of p53, the mechanism still remains to be elucidated. Here, we report that semaphorin 3F (SEMA3F), an axon guidance molecule, is involved in p53-regulated antiangiogenesis. The expression level of SEMA3F mRNA was increased by both exogenous and endogenous p53. Chromatin immunoprecipitation assay indicated that a potent p53-binding sequence in intron 1 of SEMA3F interacts with p53 and that it has a p53-responsive transcriptional activity. Overexpression of SEMA3F inhibited in vitro cell growth of the lung cancer cell line H1299. In nude mice assay, the size of the H1299 tumors expressing SEMA3F was much smaller, and they showed lesser number of blood vessels as compared with the control tumors. Moreover, tumors derived from the p53-knockdown colorectal cancer cell line LS174T displayed a remarkable enhancement of tumor vessel formation as compared with control tumors containing normal levels of p53. The expression levels of SEMA3F and neuropilin-2 (NRP2), the functional receptor for SEMA3F, in p53-knockdown LS174T tumors were lower than those in the control tumors. Adenovirus-mediated SEMA3F gene transfer induced the remarkable in vitro growth suppression of the stable transformant of H1299 cells, which express high levels of NRP2. These results suggest that p53 negatively regulates tumor vessel formation and cell growth via the SEMA3F-NRP2 pathway.

Polypeptide growth factors form a potent class of extracellular signal molecules in the regulation of cellular differentiation and proliferation. Disturbances in the expression of growth factors influence the normal pathway of differentiation and lead to cellular transformation and tumour progression. Contemporary medical studies report that various growth factors such as those for platelet-derived growth factor, vascular endothelial growth factor, epidermal growth factor, hepatocyte growth factor and insulin-like growth factor are expressed in gastroenteropancreatic neuroendocrine tumours (GEP/NET). Polypeptide growth factors have great significance in the growth, progression and development of metastases by various tumours. We describe the role of growth factors in GEP/NET on the basis of the available reports of medical research.

Although axon guidance molecules play critical roles in neural circuit formation during development, their roles in the adult circuit are not well understood. In this study we examined the expression patterns of Semaphorin 3E (Sema3E), a member of the semaphorin family, in the mature neocortices of monkeys and mice by in situ hybridization (ISH). We found that Sema3E mRNA is highly specific to layer VI throughout the macaque monkey neocortex. We further examined the ratio of Sema3E+ cells among the layer VI excitatory neurons in areas M1, S1, TE, and V1 by fluorescence double ISH, using the vesicular glutamate transporter 1 (VGluT1) gene as a specific marker for excitatory neurons. Among these areas, 34-63% of the VGluT1+ neurons expressed Sema3E mRNA. In the mouse cortex, two significant differences were observed in the pattern of Sema3E mRNA distribution. 1) Sema3E mRNA was expressed in layer Vb, in addition to layer VI in mice. 2) A subset of GABAergic interneurons expressed Sema3E mRNA in mice. By an in vitro binding experiment, we provide evidence that Plexin D1 is the specific receptor for Sema3E. Plexin D1 mRNA was preferentially expressed in layers II-V in both monkey and mouse cortices. The detailed lamina analysis by double ISH, however, revealed that Plexin D1 mRNA is expressed in layers II-Va, but not in layer Vb in the mouse cortex. Thus, the Plexin D1 and Sema3E mRNAs exhibit conserved complementary lamina patterns in mice and monkeys, despite the species differences in the pattern of each gene., (2006 Wiley-Liss, Inc.)

Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play important roles in mitogenesis and chemotaxis of endothelial cells. In normal human skin, VEGF is expressed and secreted by epidermal keratinocytes. Emerging data suggest that keratinocyte-derived VEGF targets other cell types besides the dermal endothelial cells. We have recently showed that keratinocytes from human normal skin expressed all five known VEGF receptors and co-receptors (neuropilin 1 and 2). To define the functional significance of VEGFR-2 in epidermis, we examined its role in a keratinocyte cell line, HaCaT cells, in response to VEGF treatment. Expression of VEGFR-2 on HaCaT cells was confirmed at both RNA and protein levels and was regulated by VEGF165 treatment. Treatment of HaCaT cells with VEGF165 induced tyrosine-autophosphorylation of VEGFR-2 and phosphorylation of PLC-gamma and p44/42 MAPK in a time-dependent manner. Preincubation with a neutralizing antibody for VEGFR-2 (MAB3571) completely abrogated these phosphorylation effects. Furthermore, VEGF165 stimulated proliferation and migration of HaCaT cells, and this effect was significantly blocked by a pretreatment with MAB3571. Neutralizing VEGFR-2 in HaCaT cells increased cell adhesion during culture. Our results suggest that VEGFR-2 expressed on HaCaT cells plays a crucial role in VEGF-mediated regulation of cell activity.