Expression of NRP-1 and NRP-2 in Endometrial Cancer.

BACKGROUND: Neuropilins (NRPs) participate in many processes related to cancer development such as angiogenesis, lymphangiogenesis and metastasis. Although endometrial cancer is one of the most common gynecological cancers, it has not been studied in terms of NRPs expression. OBJECTIVE: The aim of this study was to investigate the potential utility of NRPs as important factors in the diagnosis and treatment of endometrial cancer. METHODS: Our study consisted of 45 women diagnosed with endometrial cancer at the following degrees of histological differentiation: G1, 17; G2, 15; G3, 13 cases. The control group included 15 women without neoplastic changes. The immunohistochemical reactions were evaluated using light microscopy. RESULTS: We did not detect the expression of NRP-1 and NRP-2 in the control group. NRP-1 expression was found exclusively in cancer cells. It was higher in G2 and G3 and reached about 190% of G1. NRP-2 expression was observed in the endothelium and was similar across all three cancer grades. In cancer cells, NRP-2 expression increased with the degree of histological differentiation. CONCLUSION: NRP1 and NRP2 are candidates for complementary diagnostic molecular markers and promising new targets for molecular, personalized anticancer therapies.

LncRNA XIST facilitates proliferation and epithelial-mesenchymal transition of colorectal cancer cells through targeting miR-486-5p and promoting neuropilin-2.

This study was designed to acertain whether the long noncoding RNA (lncRNA) X-inactive specific transcript (XIST)/miR-486-5p/neuropilin-2 (NRP-2) pathway might promote the viability and epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells. In this investigation, we included 317 pathologically confirmed CRC patients and purchased several human CRC cells (i.e. HCT116, HT29, SW620, and SW480). Moreover, pcDNA3.1-XIST, si-XIST, miR-486-5p mimic, miR-486-5p inhibitor, and pcDNA3.1-NRP-2 were transfected into the CRC cells. And the dual-luciferase reporter gene assay managed to verify the targeted relationships among XIST, miR-486-5p, and NRP-2. Ultimately, the MTT assay, flow cytometry, colony formation assay, and transwell assay were carried out to assess the influence of XIST, miR-486-5p, and NRP-2 on the proliferation, apoptosis, migration, and invasion of CRC cells. Our study results demonstrated that CRC tissues and cells were detected with significantly elevated XIST and NRP-2 expressions as well as markedly reduced miR-486-5p expression when compared with normal tissues and cells (all p < 0.05). Besides this, the highly expressed XIST and NRP-2, as well as the lowly expressed miR-486-5p all could substantially encourage proliferation and EMT of CRC cells and simultaneously restrict apoptosis of the cells ( p < 0.05). Moreover, XIST was found to directly target miR-486-5p, and NRP-2 was directly targeted and modulated by miR-486-5p. Finally, CRC cells of the miR-NC + pcDNA3.1-NRP-2 groups showed stronger proliferation, viability, and EMT than those of miR-NC and miR-486-5p mimic groups ( p < 0.05). In conclusion, the XIST/miR-486 -5p/NRP-2 axis appeared to participate in the progression of CRC, which could assist in developing efficacious therapies for CRC.

Refuting the hypothesis that semaphorin-3f/neuropilin-2 exclude blood vessels from the cap mesenchyme in the developing kidney.

BACKGROUND: During murine kidney development, new cortical blood vessels form and pattern in cycles that coincide with cycles of collecting duct branching and the accompanying splitting of the cap mesenchyme (nephron progenitor cell populations that "cap" collecting duct ends). At no point in the patterning cycle do blood vessels enter the cap mesenchyme. We hypothesized that the exclusion of blood vessels from the cap mesenchyme may be controlled, at least in part, by an anti-angiogenic signal expressed by the cap mesenchyme cells. RESULTS: We show that semaphorin-3f (Sema3f), a known anti-angiogenic factor, is expressed in cap mesenchymal cells and its receptor, neuropilin-2 (Nrp2), is expressed by newly forming blood vessels in the cortex of the developing kidney. We hypothesized that Sema3f/Nrp2 signaling excludes vessels from the cap mesenchyme. Genetic ablation of Sema3f and of Nrp2, however, failed to result in vessels invading the cap mesenchyme. CONCLUSIONS: Despite complementary expression patterns, our data suggest that Sema3f and Nrp2 are dispensable for the exclusion of vessels from the cap mesenchyme during kidney development. These results should provoke additional experiments to ascertain the biological significance of Sema3f/Nrp2 expression in the developing kidney. Developmental Dynamics 246:1047-1056, 2017. © 2017 Wiley Periodicals, Inc.

The impact of immunohistochemical staining with ezrin-carbonic anhydrase IX and neuropilin-2 on prognosis in patients with metastatic renal cell cancer receiving tyrosine kinase inhibitors.

The identification of prognostic factors in patients with renal cell carcinoma (RCC) represents an area of increasing interest. In this retrospective study, we evaluated the prognostic role of carbonic anhydrase-IX, ezrin, and neuropilin in metastatic RCC patients. The expression of several biomarkers were measured by immunohistochemistry (IHC) in 45 patients with advanced stage RCC treated with second-line tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factor (VEGF) after failure of interferon-alpha between January 2007 and June 2012. Kaplan-Meier curves and log-rank tests were used for analysis of progression-free survival (PFS) and overall survival (OS), and a multivariate Cox proportional hazard model was employed to identify factors with an independent effect on the survival. Age, ezrin and neuropilin-2 overexpression were found to be statistically significant factors (P < 0.05) for PFS in the univariate analysis. Ezrin and neuropilin-2 overexpression, hemoglobin and albumin level were statistically significant factors (P < 0.05) for OS in the univariate analysis. Multivariate analysis revealed that low expression of ezrin and neuropilin-2 was an independent prognostic factor for PFS and OS. The median PFS was 4 months for patients overexpressing neuropilin-2 versus 11 months for those with lower expression of neuropilin-2 (p = 0.033). The median OS was longer in patients with low levels of neuropilin-2 expression (26 months) compared to patients overexpressing neuropilin-2 (13 months) (p = 0.023). Increased expression of ezrin was associated with poor prognosis in patients treated with TKIs targeting VEGF (PFS, 3 vs 7 months; p = 0.012). High ezrin expression was associated with shorter OS (p = 0.009). This is the first study in the literature showing that neuropilin-2 and ezrin are related with prognosis in patients with advanced RCC.

Expression patterns of Sema3F, PlexinA4, -A3, Neuropilin1 and -2 in the postnatal mouse molar suggest roles in tooth innervation and organogenesis.

OBJECTIVE: Semaphorins form a family of axon wiring molecules but still little is known about their role in tooth formation. A class 3 semaphorin, Semaphorin3F (Sema3F), besides acting as a chemorepellant for different types of axons, controls a variety of non-neuronal developmental processes. MATERIALS AND METHODS: Cellular mRNA expression patterns of Sema3F as well as neuropilin 1 (Npn1), neuropilin 2 (Npn2), plexinA3 and plexinA4 receptors were analyzed by sectional in situ hybridization in the mouse molar tooth during postnatal days 0-7. The expression of the receptors was studied in PN5 trigeminal ganglia. RESULTS: Sema3F, Npn1, -2 and plexinA4 exhibited distinct, spatiotemporally changing expression patterns, whereas plexinA3 was not observed in the tooth germs. Besides being expressed in the base of the dental mesenchyme Sema3F, like plexinA4, Npn1 and -2, was present in the ameloblast cell lineage. Npn1 and Npn2 were additionally seen in the pulp horns and endothelial cells and like PlexinA4 in the developing alveolar bone. Npn1, plexinA3 and -A4 were observed in trigeminal ganglion neurons. CONCLUSIONS: Sema3F may act as a tooth target-derived axonal chemorepellant controlling establishment of the tooth nerve supply. Furthermore, Sema3F, like Npn1, -2 and plexinA4 may serve non-neuronal functions by controlling the development of the ameloblast cell lineage. Moreover, Npn1 and Npn2 may regulate dental vasculogenesis and, together with PlexinA4, alveolar bone formation. Further analyses such as investigation of transgenic mouse models will be required to elucidate in vivo signaling functions of Sema3F and the receptors in odontogenesis.

Integration of genotypic and phenotypic screening reveals molecular mediators of melanoma-stromal interaction.

Tumor-endothelium interactions are critical for tumor survival and metastasis. Melanomas can rapidly metastasize early in tumor progression, but the dependence of this aggressive behavior on tumor-stromal interaction is poorly understood. To probe the mechanisms involved, we developed a heterotypic coculture methodology, allowing simultaneous tracking of genomic and phenotypic changes in interacting tumor and endothelial cells in vitro. We found a dramatic rearrangement of endothelial cell networks into patterns reminiscent of vascular beds, even on plastic and glass. Multiple genes were upregulated in the process, many coding for cell surface and secreted proteins, including Neuropilin-2 (NRP2). A critical role of NRP2 in coordinated cell patterning and growth was confirmed using the coculture system. We conclude that NRP2 represents an important mediator of melanoma-endothelial interactions. Furthermore, the described methodology represents a powerful yet simple system to elucidate heterotypic intercellular interactions mediating diverse physiological and pathological processes.

Maternal dietary restriction and selenium supply alters messenger ribonucleic acid expression of angiogenic factors in maternal intestine, mammary gland, and fetal jejunal tissues during late gestation in pregnant ewe lambs.

The objectives of this study were to evaluate effects of maternal dietary restriction and Se supply on angiogenic factor mRNA expression in intestinal and mammary tissues, and jejunal crypt cell proliferation and vascularity in late-term fetal intestines. In Exp. 1, pregnant ewe lambs (n = 32; initial BW = 45.6 +/- 2.3 kg) were allotted randomly to 1 of 4 treatments. Treatments (initiated d 50 +/- 5 of gestation) were control (3.5 microg of Se.kg of BW(-1).d(-1)), Se-wheat (75 microg of Se.kg of BW(-1).d(-1)), selenate (Se3; providing 75 microg of Se.kg of BW(-1).d(-1)), selenate (Se15; providing 375 microg of Se.kg of BW(-1).d(-1)). Diets (DM basis) were similar in CP (15.5%) and ME (2.68 Mcal/kg). In Exp. 2, pregnant ewe lambs (n = 36; initial BW 53.8 +/- 1.3 kg) were allotted randomly to treatments in a 2 x 2 factorial arrangement. Factors were nutrition (control, 100% of requirements vs. restricted nutrition, 60% of controls) and dietary Se (adequate Se; 6 microg of Se.kg of BW(-1).d(-1) vs. high Se; 80 microg of Se.kg of BW(-1).d(-1)). Selenium treatments were initiated 21 d before breeding, and nutritional treatments were initiated on d 64 of gestation. Diets (DM basis) were 16% CP and 2.12 Mcal/kg of ME. In Exp. 1, Se15 increased (P = 0.07) vascular endothelial growth factor (VEGF) mRNA expression, whereas Se supplementation decreased (P = 0.06) kinase insert domain receptor (KDR) mRNA in maternal mucosal scrape on d 134 of gestation. Expression of VEGF mRNA was decreased by Se (P = 0.10) in fetal jejunum. In mammary tissue, fms-related tyrosine kinase 1 and KDR mRNA were greater in Se-wheat compared with Se3, and KDR expression was increased (P = 0.10) in Se15 compared with Se3. In Exp. 2, dietary restriction increased (P < or = 0.07) expression of mRNA for VEGF, fms-related tyrosine kinase 1, KDR, neuropilin 1, neuropilin 2, and hypoxia-inducible factor 1, alpha subunit in mucosal scrapes from maternal jejunum. In fetal jejunum, soluble guanylate cyclase, was decreased (P = 0.01) by maternal dietary restriction from d 64 to 135 of gestation. Total microvascularity in fetal jejunum was reduced (P = 0.002) by maternal dietary restriction. Mammary gland expression of VEGF, neuropilin 1, angiopoietin receptor (endothelial tyrosine kinase), and endothelial nitric oxide synthase 3 increased (P < or = 0.09), whereas angiopoietin 1 decreased (P = 0.05) due to nutrient restriction. Data indicate that expression of angiogenic factors and receptors in maternal intestine, mammary gland, and fetal jejunum are responsive to maternal nutrition and likely explain observed changes in tissue vascularity.

Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development.

The lymphatic system plays a key role in tissue fluid homeostasis. Lymphatic dysfunction contributes to the pathogenesis of many human diseases, including lymphedema and tumor metastasis. However, the mechanisms regulating lymphangiogenesis remain largely unknown. Here, we show that COUP-TFII (also known as Nr2f2), an orphan member of the nuclear receptor superfamily, mediates both developmental and pathological lymphangiogenesis in mice. Conditional ablation of COUP-TFII at an early embryonic stage resulted in failed formation of pre-lymphatic ECs (pre-LECs) and lymphatic vessels. COUP-TFII deficiency at a late developmental stage resulted in loss of LEC identity, gain of blood EC fate, and impaired lymphatic vessel sprouting. siRNA-mediated downregulation of COUP-TFII in cultured primary human LECs demonstrated that the maintenance of lymphatic identity and VEGF-C-induced lymphangiogenic activity, including cell proliferation and migration, are COUP-TFII-dependent and cell-autonomous processes. COUP-TFII enhanced the pro-lymphangiogenic actions of VEGF-C, at least in part by directly stimulating expression of neuropilin-2, a coreceptor for VEGF-C. In addition, COUP-TFII inactivation in a mammary gland mouse tumor model resulted in inhibition of tumor lymphangiogenesis, suggesting that COUP-TFII also regulates neo-lymphangiogenesis in the adult. Thus, COUP-TFII is a critical factor that controls lymphangiogenesis in embryonic development and tumorigenesis in adults.

Neuropilin 2 deficiency does not affect cortical neuronal viability in response to oxygen-glucose-deprivation and transient middle cerebral artery occlusion.

Neuropilin 2 (NRP2) is a type I transmembrane protein that binds to distinct members of the class III secreted Semaphorin subfamily. NRP2 plays important roles in repulsive axon guidance, angiogenesis and vasculogenesis through partnering with co-receptors such as vascular endothelial growth factor receptors (VEGFRs) during development. Emerging evidence also suggests that NRP2 contributes to injury response and environment changes in adult brains. In this study, we examined the contribution of NRP2 gene to cerebral ischemia-induced brain injury using NRP2 deficient mouse. To our surprise, the lack of NRP2 expression does not affect the outcome of brain injury induced by transient occlusion of the middle cerebral artery (MCAO) in mouse. The cerebral vasculature in terms of the middle cerebral artery anatomy and microvessel density in the cerebral cortex of NRP2 deficient homozygous (NRP2(-/-)) mice are normal and almost identical to those of the heterozygous (NRP2(+/-)) and wild type (NRP2(+/+)) littermates. MCAO (1h) and 24h reperfusion caused a brain infarction of 23% (compared to the contralateral side) in NRP2(-/-) mice, which is not different from those in NRP2(+/- and +/+) mice at 22 and 21%, respectively (n=19, p>0.05). Correspondingly, NRP2(-/-) mouse also showed a similar level of deterioration of neurological functions after stroke compared with their NRP2(+/- and +/+) littermates. Oxygen-glucose-deprivation (OGD) caused a significant neuronal death in NRP2(-/-) cortical neurons, at the level similar to that in NRP(+/+) cortical neurons (72% death in NRP(-/-) neurons vs. 75% death in NRP2(+/+) neurons; n=4; p>0.05). Together, these loss-of-function studies demonstrated that despite of its critical role in neuronal guidance and vascular formation during development, NRP2 expression dose not affect adult brain response to cerebral ischemia.

VEGFR-2 silencing by small interference RNA (siRNA) suppresses LPA-induced epithelial ovarian cancer (EOC) invasion.

BACKGROUND: The VEGF-VEGF receptor (VEGFR) signaling axis has emerged as a promising target for cancer therapy, attributing to its vital role in tumor angiogenesis and growth. We have previously reported the regulation of epithelial ovarian cancer (EOC) invasion and migration by VEGF and the implication of VEGF-VEGFR-2 axis in lysophosphatidic acid (LPA)-induced EOC invasion. However, the expression profile of VEGF and VEGFRs in EOC, their association with tumor aggressiveness, and their regulation by LPA remain unclear. OBJECTIVES AND METHODS: In this study, we examined the expression of VEGFR-1, VEGFR-2, neuropilin-1 (NRP-1), NRP-2, VEGF(121), and VEGF(165) in established EOC cell lines and assessed their correlation with cell invasiveness. Moreover, using an ovarian cancer tissue qPCR array, we analyzed VEGFR-2 expression across a panel of 48 tissues with different disease stages and histological grades. We also tested the effect of LPA on VEGF and VEGFR-2 expression and examined whether blocking VEGFR-2 by RNA interference (RNAi) affects LPA-induced EOC invasion. RESULTS: We show that VEGF and VEGFR-2 expression correlates with cell invasiveness and VEGFR-2 expression in ovarian cancer tissues correlate with tumor grade. In addition, LPA, at 20 muM, significantly induced the expression of VEGF(121), VEGF(165), and VEGFR-2 in SKOV3 and DOV13 cells (P<0.05). VEGFR-2 small interference RNA (siRNA) transfection remarkably decreased LPA's invasion-promoting effect (P<0.001) in SKOV3 cells without significantly decreasing SKOV3 cells' basal invasiveness. In DOV13 cells, VEGFR-2 silencing significantly decreases both the basal level cell invasion and LPA's invasion promoting effect (P<0.001). CONCLUSION: These results suggest that decreasing VEGFR-2 expression by RNAi may prove to be an effective method to reduce the metastatic potential of EOC cells exposed to elevated levels of LPA.

Vascular endothelial growth factor-C protects prostate cancer cells from oxidative stress by the activation of mammalian target of rapamycin complex-2 and AKT-1.

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.

Neuropilin-2 expression in breast cancer: correlation with lymph node metastasis, poor prognosis, and regulation of CXCR4 expression.

BACKGROUND: Neuropilin-2 (Nrp2) is a receptor for vascular endothelial growth factor-C (VEGF-C), which is a well-known lymphangiogenic factor and plays an important role in lymph node metastasis of various human cancers, including breast cancer. Recently, Nrp2 was shown to play a role in cancer by promoting tumor cell metastasis. CXC chemokine receptor 4 (CXCR4) also promotes tumor metastasis. In the previous studies, we demonstrated that VEGF-C and cytoplasmic CXCR4 expressions were correlated with poorer patient prognosis (BMC Cancer 2008,8:340; Breast Cancer Res Treat 2005, 91:125-132). METHODS: The relationship between Nrp2 expression and lymph node metastasis, VEGF-C expression, CXCR4 expression, and other established clinicopathological variables (these data were cited in our previous papers), including prognosis, was analyzed in human breast cancer. Effects of neutralizing anti-Nrp2 antibody on CXCR4 expression and chemotaxis were assessed in MDA-MB-231 breast cancer cells. RESULTS: Nrp2 expression was observed in 53.1% (60 of 113) of the invasive breast carcinomas. Nrp2 expression was significantly correlated with lymph node metastasis, VEGF-C expression, and cytoplasmic CXCR4 expression. Survival curves determined by the Kaplan-Meier method showed that Nrp2 expression was associated with reduced overall survival. In multivariate analysis, Nrp2 expression emerged as a significant independent predictor for overall survival. Neutralizing anti-Nrp2 antibody blocks cytoplasmic CXCR4 expression and CXCR4-induced migration in MDA-MB-231 cells. CONCLUSION: Nrp2 expression was correlated with lymph node metastasis, VEGF-C expression, and cytoplasmic CXCR4 expression. Nrp2 expression may serve as a significant prognostic factor for long-term survival in breast cancer. Our data also showed a role for Nrp2 in regulating cytoplasmic CXCR4 expression in vitro.

VEGF receptors and neuropilins are expressed in the urothelial and neuronal cells in normal mouse urinary bladder and are upregulated in inflammation.

Recent evidence supports a role for vascular endothelium growth factor (VEGF) signaling in bladder inflammation. However, it is not clear what bladder cells are targeted by VEGF. Therefore, we determined the nature of cells responding to VEGF in normal and inflamed bladders by tagging such cells in vivo with a targeted fluorescent tracer, scVEGF/Cy, an engineered single-chain VEGF labeled with Cy5.5 dye, which identifies cells with accessible and functionally active VEGF receptors. Inflammation was induced by intravesical instillation of PAR-activating peptides or BCG. In vivo NIRF imaging with intravenously injected scVEGF/Cy revealed accumulation of the tracer in the control mouse bladder and established that inflammation increased the steady-state levels of tracer uptake. Ex vivo colocalization of Cy5.5 dye revealed that in normal and at a higher level in inflamed bladder, accumulation of scVEGF/Cy occurs in both urothelial and ganglial cells, expressing VEGF receptors VEGFR-1 and VEGFR-2, as well as VEGF coreceptors neuropilins (NRP) NRP1 and NRP2. PCR results indicate that the messages for VEGF-Rs and NRPs are present in the bladder mucosa and ChIP/QPCR analysis indicated that inflammation induced upregulation of genes encoding VEGFRs and NRPs. Our results strongly suggest new and blossoming VEGF-driven processes in bladder urothelial cells and ganglia in the course of inflammation. We expect that molecular imaging of the VEGF pathway in the urinary tract by receptor-mediated cell tagging in vivo will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of bladder-targeting drugs and treatments.

Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells.

Neuropilin (NRP) receptors and their class 3 semaphorin (SEMA3) ligands play well-established roles in axon guidance, with loss of NRP1, NRP2, SEMA3A or SEMA3F causing defasciculation and errors in growth cone guidance of peripherally projecting nerves. Here we report that loss of NRP1 or NRP2 also impairs sensory neuron positioning in the mouse head, and that this defect is a consequence of inappropriate cranial neural crest cell migration. Specifically, neural crest cells move into the normally crest-free territory between the trigeminal and hyoid neural crest streams and recruit sensory neurons from the otic placode; these ectopic neurons then extend axons between the trigeminal and facioacoustic ganglia. Moreover, we found that NRP1 and NRP2 cooperate to guide cranial neural crest cells and position sensory neurons; thus, in the absence of SEMA3/NRP signalling, the segmentation of the cranial nervous system is lost. We conclude that neuropilins play multiple roles in the sensory nervous system by directing cranial neural crest cells, positioning sensory neurons and organising their axonal projections.

Myeloid leukemias express a broad spectrum of VEGF receptors including neuropilin-1 (NRP-1) and NRP-2.

Vascular endothelial growth factor (VEGF) is produced in neoplastic cells in various myeloid neoplasms and may act as an autocrine growth-regulator. We have examined the expression of five VEGF receptors (VEGR1/Flt-1, VEGFR2/KDR, Flt-4, neuropilin-1 = NRP-1, NRP-2) in leukemic cells obtained from patients with acute myeloid leukemia (n = 28), chronic myeloid leukemia (n = 14), chronic eosinophilic leukemia (n = 3), chronic myelomonocytic leukemia (n = 9), or mast cell leukemia/systemic mastocytosis (n = 3) as well as in respective cell lines. Expression of VEGFR mRNA was analyzed by RT-PCR, and expression of VEGFR protein by immunocytochemistry. In most patients, leukemic cells expressed NRP-1 mRNA and NRP-2 mRNA independent of the type of disease. By contrast, transcripts for Flt-1, KDR, and Flt-4 were expressed variably without a clear correlation to the type of leukemia. Expression of VEGF receptors was also demonstrable at the protein level in all cases tested. In conclusion, neoplastic cells in myeloid leukemias frequently express VEGFR including NRP-1 and NRP-2.

Expression and functions of the vascular endothelial growth factors and their receptors in human basophils.

Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of vascular endothelial growth factors (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (121, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 +/- 10.8 pg/10(6) cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10-500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.

Expression of semaphorins, vascular endothelial growth factor, and their common receptor neuropilins and alleic loss of semaphorin locus in epithelial ovarian neoplasms: increased ratio of vascular endothelial growth factor to semaphorin is a poor prognostic factor in ovarian carcinomas.

Semaphorins (SEMAs) compete with vascular endothelial growth factor (VEGF) for receptor neuropilin 1 (NP1) and 2 (NP2) and suppress angiogenesis. To clarify the involvement of SEMA and VEGF in the development and progression of ovarian carcinoma, we analyzed the immunohistochemical expression of SEMA, VEGF, NP1, and NP2 in 105 epithelial ovarian tumors. In addition, loss of heterozygosity at SEMA gene loci was examined. Strong expression of SEMA was found in 48% of benign, 33% of borderline tumors, and 13% of carcinomas (P < .05). Positivity for SEMA was significantly decreased in stage IV carcinomas and the expression of SEMA was significantly lower in peritoneal metastases than in primary lesions. Expression of SEMA showed a weak inverse correlation with microvessel density, but the correlation was not statistically significant. Loss of heterozygosity at SEMA3B or SEMA3F was demonstrated in none of the benign tumors, 8% of borderline tumors, and 29% of carcinomas. Expression of NP1 and NP2 was significantly higher in carcinomas than in benign tumors (P < .0001 and .0002, respectively). Patients with ovarian carcinoma with a high VEGF/SEMA ratio showed poorer survival than those with a low VEGF/SEMA ratio (P = .005). Decreased expression of SEMA and increased expression of NP1 and NP2 are characteristics of ovarian carcinomas, and loss of SEMA expression may play an important role in ovarian carcinoma progression. A high VEGF/SEMA ratio has adverse prognostic significance in patients with ovarian carcinoma.

Changes in protein expression in the rat medial vestibular nuclei during vestibular compensation.

The molecular mechanisms of neural and synaptic plasticity in the vestibular nuclei during 'vestibular compensation', the behavioural recovery that follows deafferentation of one inner ear, are largely unknown. In this study we have used differential proteomics techniques to determine changes in protein expression in ipsi-lesional and contra-lesional medial vestibular nuclei (MVN) of rats, 1 week after either sham surgery or unilateral labyrinthectomy (UL). A systematic comparison of 634 protein spots in two-dimensional electrophoresis gels across five experimental conditions revealed 54 spots, containing 26 proteins whose level was significantly altered 1 week post-UL. The axon-guidance-associated proteins neuropilin-2 and dehydropyriminidase-related protein-2 were upregulated in the MVN after UL. Changes in levels of further specific proteins indicate a coordinated upregulation of mitochondrial function, ATP biosynthesis and phosphate metabolism in the vestibular nuclei 1 week post-UL. These may reflect the metabolic energy demands of processes such as gliosis, neuronal outgrowth and synaptic remodelling that occur after UL. Our findings suggest novel roles for axon elaboration and guidance molecules, as well as mitochondrial and metabolic regulatory proteins, in the post-lesional physiology of the MVN during vestibular system plasticity.

The preserved expression of neuropilin (NRP) 1 contributes to a better prognosis in colon cancer.

Vascular endothelial growth factor A (VEGF-A) plays an essential role in tumor progression through stromal neovascularization in malignant solid tumors. Neuropilin (NRP) is considered to be the specific receptor for limited types of VEGF-A isoform, VEGF165. The clinicopathological implications of NRP are not well understood in colon cancer, while almost all colon cancers overexpressed VEGF-A. We examined the expression levels of NRP1 and NRP2 genes in 54 colon cancer cases and paired extraneoplastic tissue with quantitative real-time polymerase chain reaction. The gene expression levels of NRP1 in the tumor (0.431+/-0.583) were significantly decreased compared to those in the extraneoplastic tissue (0.754+/-0.799) (paired t-test, p=0.0208). On the other hand, the gene expression levels of NRP2 in the tumor (0.763+/-0.791) were not decreased compared to those in the extraneoplastic tissue (0.508+/-0.386) (paired t-test, p=0.0511). Twenty cases, with preserved expression of the NRP1 gene in the tumor, showed a better prognosis as compared to the 34 cases with decreased NRP1 expression (p=0.0258, log-rank test). No significant relationship was noted between NRP2 gene expression and prognosis. The results suggested that preserved NRP1 expression provides colon cancer patients with a better prognosis.

Repulsion and attraction of axons by semaphorin3D are mediated by different neuropilins in vivo.

Class 3 semaphorins are known to repel and/or sometimes attract axons; however, their role in guiding developing axons in the CNS in vivo is still essentially unknown. We investigated the role of Semaphorin3D (Sema3D) in the formation of the early axon pathways in the zebrafish CNS. Morpholino knock-down shows that Sema3D is essential for the correct formation of two early axon pathways. Sema3D appears to guide axons of the nucleus of the medial longitudinal fasciculus (nucMLF) by repulsion and modulation of fasciculation. In contrast, Sema3D appears to be attractive to telencephalic neurons that form the anterior commissure (AC). Knock-down of Neuropilin-1A (Npn-1A) phenocopied the effects of Sema3D knock-down on the nucMLF axons, and knock-down of either Npn-1A or Npn-2B phenocopied the defects of the AC. Furthermore, simultaneous partial knock-down experiments demonstrated genetic interactions among Sema3D, Npn-1A, and Npn-2B. Together, these data support the hypothesis that Sema3D may act as a repellent through receptors containing Npn-1A and as an attractant via receptors containing Npn-1A and Npn-2B.