Expression of CD304/neuropilin-1 in adult b-cell lymphoblastic leukemia/lymphoma and its utility for the measurable residual disease assessment.

INTRODUCTION: Many new markers are being evaluated to increase the sensitivity and applicability of multicolor flow cytometry (MFC)-based measurable residual disease (MRD) monitoring. However, most of the studies are limited to childhood B-cell lymphoblastic leukemia/lymphoma (B-ALL), and reports in adult B-ALL are extremely scarce and limited to small cohorts. We studied the expression of CD304/neuropilin-1 in a large cohort of adult B-ALL patients and evaluated its practical utility in MFC-based MRD analysis. METHODS: CD304 was studied in blasts from adult B-ALL patients and normal precursor B cells (NPBC) from non-B-ALL bone marrow samples using MFC. CD304 expression intensity and pattern were studied with normalized-mean fluorescent intensity (nMFI) and coefficient of variation of immunofluorescence (CVIF), respectively. MFC-based MRD was performed at end of induction (EOI; day-35), end of consolidation (EOC; day 78-80), and subsequent follow-up (SFU) time points. RESULTS: CD304 was positive in 120/214(56.07%) and was significantly associated with BCR-ABL1 fusion (P = .001). EOI-MRD and EOC-MRD were positive in 129/214(60.3%) and 50/81(61.72%), respectively. CD304 was positive in a significant percentage of EOI (48%, 62/129) and EOC (52%, 26/50) MRD-positive B-ALL samples. Its expression was retained, lost, and gained in 73.7%, 26.3%, and 11.3% of EOI-MRD and 85.7%, 14.3%, and none of EOC-MRD samples, respectively. Low-level MRD (<0.01%) was detectable in 34 of all (EOI + EOC + SFU = 189) MRD-positive samples, and CD304 was found useful in 50% of these samples. CONCLUSION: CD304 is commonly expressed in adult B-ALL and clearly distinguish B-ALL blasts from normal precursor B cells. It is a stable MRD marker and distinctly useful in the detection of MFC-based MRD monitoring, especially in high-sensitivity MRD assay.

Probing Membrane Protein Association Using Concentration-Dependent Number and Brightness.

We introduce concentration-dependent number and brightness (cdN&B), a fluorescence fluctuation technique that can be implemented on a standard confocal microscope and can report on the thermodynamics of membrane protein association in the native plasma membrane. It uses transient transfection to enable measurements of oligomer size as a function of receptor concentration over a broad range, yielding the association constant. We discuss artifacts in cdN&B that are concentration-dependent and can distort the oligomerization curves, and we outline procedures that can correct for them. Using cdN&B, we characterize the association of neuropilin 1 (NRP1), a protein that plays a critical role in the development of the embryonic cardiovascular and nervous systems. We show that NRP1 associates into a tetramer in a concentration-dependent manner, and we quantify the strength of the association. This work demonstrates the utility of cdN&B as a powerful tool in biophysical chemistry.

Semaphorin 3A Inhibits Nerve Regeneration During Early Stage after Inferior Alveolar Nerve Transection.

Neuroma formation at sites of injury can impair peripheral nerve regeneration. Although the involvement of semaphorin 3A has been suggested in neuroma formation, this detailed process after injury is not fully understood. This study was therefore undertaken to examine the effects of semaphorin 3A on peripheral nerve regeneration during the early stage after injury. Immunohistochemistry for semaphorin 3A and PGP9.5, a general neuronal marker, was carried out for clarify chronological changes in their expressions after transection of the mouse inferior alveolar nerve thorough postoperative days 1 to 7. At postoperative day 1, the proximal stump of the damaged IAN exhibited semaphorin 3A, while the distal stump lacked any immunoreactivity. From this day on, its expression lessened, ultimately disappearing completely in all regions of the transected inferior alveolar nerve. A local administration of an antibody to semaphorin 3A into the nerve transection site at postoperative day 3 inhibited axon sprouting at the injury site. This antibody injection increased the number of trigeminal ganglion neurons labeled with DiI (paired t-test, p < 0.05). Immunoreactivity of the semaphorin 3A receptor, neuropilin-1, was also detected at the proximal stump at postoperative day 1. These results suggest that nerve injury initiates semaphorin 3A production in ganglion neurons, which is then delivered through the nerve fibers to the proximal end, thereby contributes to the inhibition of axonal sprouting from the proximal region of injured nerves in the distal direction. To our knowledge, this is the first report to reveal the involvement of Sema3A in the nerve regeneration process at its early stage.

Differential expression of CD73, CD86 and CD304 in normal vs. leukemic B-cell precursors and their utility as stable minimal residual disease markers in childhood B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers differentially expressed on normal BCP and leukemic blasts is required. METHODS: Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values. RESULTS: CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCP-ALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (p = 0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (p = 0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of +24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases. CONCLUSIONS: Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by flow cytometry.

Antiangiogenic agent sunitinib induces epithelial to mesenchymal transition and accelerates motility of colorectal cancer cells.

Although vascular endothelial growth factor receptor (VEGF-R)-targeted antiangiogenic agents are important treatment for a number of human malignancies, there is accumulating evidence that the therapies may promote disease progression, such as invasion and metastasis. How tumors become to promote their evasiveness remains fully uncertain. One of possible mechanisms for the adaptation may be a direct effect of VEGF-R inhibitors on tumor cells expressing VEGF-R. To elucidate a direct effect of VEGF-R-targeting drug (sunitinib), we established a human colorectal cancer cell model adapted to sunitinib. The sunitinib-conditioned cells showed a significant increase in cellular motility and migration activities, compared to the vehicle-treated control cells. Consistent with the phenotype, the sunitinib-conditioned cells decreased the expression levels of E-cadherin (an epithelial marker), while significantly increased the levels of Slug and Zeb1 (mesenchymal markers). Expression profiles of VEGF-R in the sunitinib-conditioned cells showed that only neuropilin-1 (NRP1) expression was significantly increased among all VEGF-R tested. Blockade of NRP1 using its antagonist clearly repressed the migration activation in sunitinib-conditioned cells, but not in the control cells. These results suggest that inhibition of VEGF-R on colorectal cancer cells can drive the epithelial-mesenchymal transition, leading to activation of cell motility in an NRP1-dependent manner. J. Med. Invest. 64: 250-254, August, 2017.

Biodistribution and evaluation of 131 I-labeled neuropilin-binding peptide for targeted tumor imaging.

Neuropilin-1 (NRP-1) is overexpressed in several kinds of cancer cell and contributes to tumor aggressiveness. Recently, the arginine/lysine-rich peptide with C-terminal motifs (R/K)XX(R/K) indicated promising penetrating and transporting capability into NRP-1 positive cancer cells. In the present study, we describe a 131 I-labeled C-end rule motif peptide conjugate, Tyr-tLyp-1, for NRP-1 positive tumor targeting and imaging properties. Briefly, a truncated Lyp-1 peptide was designed to expose its C-end motif and conjugated to tyrosine for radiolabeling after structural modification. The peptide indicated specific binding to A549 cancer cells at 2 µM concentration, and its binding was dependent on NRP-1 expression and could be inhibited by other NRP-1-binding peptides. In vivo imaging of 131 I-labeled Tyr-tLyp-1peptide showed that a subcutaneous A549 xenograft tumor could be visualized using a SPECT/CT scanner. The tumor uptake of 131 I-Tyr-tLyp-1 was 4.77 times higher than the uptake in muscles by SPECT/CT software quantification at 6 h post injection. Together, this study indicated that truncated Lyp-1 peptide could specifically localize in NRP-1 positive tumors and successfully mediate the 131 I radionuclide diagnosis, indicating promising targeted imaging capability for NRP-1 positive tumors. Copyright © 2016 John Wiley & Sons, Ltd.

Fetal liver hematopoietic stem cell niches associate with portal vessels.

Whereas the cellular basis of the hematopoietic stem cell (HSC) niche in the bone marrow has been characterized, the nature of the fetal liver niche is not yet elucidated. We show that Nestin(+)NG2(+) pericytes associate with portal vessels, forming a niche promoting HSC expansion. Nestin(+)NG2(+) cells and HSCs scale during development with the fractal branching patterns of portal vessels, tributaries of the umbilical vein. After closure of the umbilical inlet at birth, portal vessels undergo a transition from Neuropilin-1(+)Ephrin-B2(+) artery to EphB4(+) vein phenotype, associated with a loss of periportal Nestin(+)NG2(+) cells and emigration of HSCs away from portal vessels. These data support a model in which HSCs are titrated against a periportal vascular niche with a fractal-like organization enabled by placental circulation.

Increased expression of neuropilin 1 in melanoma progression and its prognostic significance in patients with melanoma.

Neuropilin 1 (NRP1), a receptor of vascular endothelial growth factor (VEGF), promotes angiogenesis, tumor growth, tumor invasion and metastasis. However, the function of NRP1 in melanoma progression, as well as the effect of NRP1 expression on the prognosis of patients with melanoma remains unknown. In the present study, NRP1 expression was examined in 460 cases of melanocytic lesions (28 common nevi, 51 dysplastic nevi, 250 primary melanoma and 131 metastatic melanoma) at different stages, using a tissue microarray. The correlation of NRP1 expression with melanoma progression, and its prognostic value in patients with melanoma was examined. In addition, the correlation between matrix metalloproteinase 2 (MMP2) and NRP1 expression in patients with melanoma was analyzed. The results demonstrated that NRP1 expression was significantly increased in primary (56%) and metastatic melanoma (62%), compared with common nevi (11%) and dysplastic nevi (24%). Notably, increased NRP1 expression was correlated with a poorer overall, and disease-specific, 10-year survival (P=0.03 and P=0.002, respectively). Multivariate Cox regression analyses indicated that NRP1 is an independent prognostic marker for melanoma. Furthermore, a significant positive correlation between NRP1 and MMP2 expression in melanoma biopsies was observed, and their concomitant expression was closely correlated with melanoma patient survival, further supporting the hypothesis that the expression of NRP1 is associated with melanoma invasion and metastasis. In conclusion, increased NRP1 expression is associated with disease progression and reduced survival in patients with melanoma, and is a promising prognostic molecular marker for this disease.

Increased expression of neuropilin 1 is associated with epithelial ovarian carcinoma.

Neuropilin 1 (NRP1) is a transmembrane glycoprotein, which interacts with vascular endothelial growth factor to prevent tumor cell apoptosis and to regulate angiogenesis. However, the precise role of NRP1 in epithelial ovarian carcinoma (EOC) remains to be elucidated. The present study aimed to determine the association between NRP1 and EOC. The expression of NRP1 in ovarian cancer and normal ovarian epithelial tissues was investigated by immunofluorescence, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. The association between the expression of NRP1 with the development of ovarian cancer, clinicopathological characteristics and survival were also analyzed. The results from immunofluorescence, RT-qPCR and western blot analysis demonstrated that NRP1 exhibited significant upregulation in EOC compared with normal ovarian epithelial specimens (P<0.05). The positive expression of NRP1 was higher in cancer tissues at an advanced International Federation of Gynecology and Obstetrics stage, and in cancer tissues with lymph node metastasis and distant metastasis compared with that in cancer tissues without lymph node or distant metastasis (P<0.05). Higher NRP1 expression strongly predicted a shorter survival time (P<0.001). The present findings suggested that increased NRP1 expression may be associated with the development of EOC. Therefore, NRP1 could be used as a valuable prognostic marker as well as a potential molecular therapy target for ovarian cancer patients.

Cilengitide downmodulates invasiveness and vasculogenic mimicry of neuropilin 1 expressing melanoma cells through the inhibition of αvß5 integrin.

During melanoma progression, tumour cells show increased adhesiveness to the vascular wall, invade the extracellular matrix (ECM) and frequently form functional channels similar to vascular vessels (vasculogenic mimicry). These properties are mainly mediated by the interaction of integrins with ECM components. Since we had previously identified neuropilin 1 (NRP-1), a coreceptor of vascular endothelial growth factor A (VEGF-A), as an important determinant of melanoma aggressiveness, aims of this study were to identify the specific integrins involved in the highly invasive phenotype of NRP-1 expressing cells and to investigate their role as targets to counteract melanoma progression. Melanoma aggressiveness was evaluated in vitro as cell ability to migrate through an ECM layer and to form tubule-like structures using transfected cells. Integrins relevant to these processes were identified using specific blocking antibodies. The αvß5 integrin was found to be responsible for about 80% of the capability of NRP-1 expressing cells to adhere on vitronectin. In these cells αvß5 expression level was twice higher than in low-invasive control cells and contributed to the ability of melanoma cells to form tubule-like structures on matrigel. Cilengitide, a potent inhibitor of αν integrins activation, reduced ECM invasion, vasculogenic mimicry and secretion of VEGF-A and metalloproteinase 9 by melanoma cells. In conclusion, we demonstrated that ανß5 integrin is involved in the highly aggressive phenotype of melanoma cells expressing NRP-1. Moreover, we identified a novel mechanism that contributes to the antimelanoma activity of the αv integrin inhibitor cilengitide based on the inhibition of vasculogenic mimicry.

SPECT and near-infrared fluorescence imaging of breast cancer with a neuropilin-1-targeting peptide.

Breast cancer is the most common malignant cancer and is the leading cause of cancer death among females. Molecular imaging is a promising approach for the early detection and staging of breast cancer as well as for assessing therapeutic responses. Tumor-targeting peptides are effective targeting vehicles for molecular imaging. Here, we identified a breast cancer-targeting peptide CLKADKAKC (CK3) contains a cryptic C-end rule motif that may mediate its binding to neuropilin-1 (NRP-1), an attractive therapeutic target which expression was associated with poor outcome of the patients with breast cancer. Phage CK3 bound to NRP-1-positive breast cancer cells, which could be inhibited by peptide CK3 in a dose-dependent manner or by knock-down NRP-1 expression. Consistently, NRP-1 overexpression in cells increased the binding of phage CK3. Furthermore, peptide CK3 co-localized with NRP-1. Importantly, unlike previously reported NRP-1-targeting peptides with exposed C-end rule motifs, peptide CK3 did not penetrate into lungs and heart in vivo, which could make it more clinically applicable. Single-photon emission CT (SPECT) and near-infrared fluorescence (NIRF) imaging showed enrichment of peptide CK3 to the xenograft tumors in nude mice. In conclusion, as a novel NRP-1-targeting peptide, peptide CK3 could be used for breast cancer molecular imaging, which may represent a new avenue for breast cancer diagnostics, staging and assessments of therapeutic response.

NRP-1 expression in bladder cancer and its implications for tumor progression.

Neuropilin-1 (NRP-1) overexpression has been reported in a variety of human cancers. However, the role of NRP-1 in bladder cancer (BC) remains unclear. The aim of present study was to analyze NRP-1 protein expression in BC tissues and to assess its prognostic significance for BC. NRP-1 messenger ribonucleic acid (mRNA) and protein expression were determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunohistochemistry in specimens of primary cancer and their adjacent noncancerous tissues in BC patients. Additionally, NRP-1 protein expression in 139 archived paraffin-embedded BC samples was analyzed by immunohistochemistry and correlated with clinicopathological characteristics and survival. Student's t test, Spearman's rank correlation, Kaplan-Meier plots, and Cox's proportional hazards regression model were used to analyze the data. By qRT-PCR and immunohistochemistry, the levels of NRP-1 mRNA and protein were significantly higher in BC, compared to that in adjacent noncancerous tissues (P<0.001). High expression of NRP-1 was significantly associated with histologic grade (P=0.016) and tumor stage (P=0.001). Multivariate analysis showed that high expression of NRP-1 was an independent prognostic factor for overall survival. Our study suggests that overexpression of NRP-1 may play an important role in the progression of BC, and NRP-1 expression may serve as a biomarker for poor prognosis for BC.

Neuropilin-1 expression in squamous cell carcinoma of the oesophagus.

OBJECTIVES: The development of new therapeutic targets is needed to change the current low survival rates of cancer of the oesophagus. In some clinical trials, angiogenic inhibitors, including those targeting the vascular endothelial growth factor (VEGF) and its receptors, have proven efficacious. In concert with this, neuropilin-1 (NRP1), a coreceptor for VEGF, is expressed by many tumours and may be related to their progression. This study aimed to assess the expression and prognostic value of fNRP1 in primary squamous cell carcinoma (SCC) of the oesophagus. METHODS: The expression of NRP1 receptors was assessed in 60 samples of resected oesophageal SCC and adjacent normal mucosa by western blotting, immunostaining and real-time quantitative PCR (qPCR). Furthermore, the relationship between NRP1 and the clinicopathological parameters was investigated. RESULTS: NRP1 staining was limited within normal tissues of the oesophagus, while it was prominent in tumour cells and vasculature. Overexpression of NRP1 receptors (3.6 ± 0.48-folds) was apparent in 81.7% specimens (n = 60, P = 0.0001). qPCR consistently revealed parallel NRP1-mRNA overexpression (3.7 ± 3.7-folds) (n = 16, P = 0.02). A higher molecular weight-modified NRP1 (mNRP1) species was identified in a large proportion of the tumour specimens (85%), accounting for 71.51 ± 20.6% of their total NRP1. Overexpression of tumour NRP1 was positively correlated with deeper invasion into the oesophageal wall (P = 0.05) though mNRP1-positive tumour populations were significantly associated with less lymph node metastasis (P = 0.036) and better prognostic tumour-node-metastasis stage (P = 0.037) than mNRP1 negative tumours. CONCLUSIONS: NRP1 overexpression in oesophageal SCC may contribute to local tumour invasiveness but the presence of the mNRP1 subtype correlates with less lymph node metastasis and better prognostic stage, suggesting that the balance between modified and unmodified NRP1 might be important for determining invasion potential.

Prognostic implication of neuropilin-1 upregulation in human nasopharyngeal carcinoma.

OBJECTIVE: As a receptor for both vascular endothelial growth factors and semaphorin, neuropilin-1 (NRP-1) is reported to be up-regulated in cells of several cancers. However, its roles in human nasopharyngeal carcinoma (NPC) are still unclear. Therefore, the goal of this study was to investigate the expression pattern of NRP-1 in NPC tissues, to clarify the clinical significance of NRP-1 expression in NPC as well as the potential prognostic implication of NRP-1 expression. METHODS: Immunohistochemistry was performed to detect the expression of NRP-1 in tumor tissue samples from 266 NPC patients. The association of NRP-1 protein expression with the clinicopathological characteristics and the prognosis of NPC were subsequently assessed. RESULTS: Immunohistochemical analysis showed that 176 of 266 (66.17%) paraffin-embedded archival NPC biopsies showed high expression of NRP-1, but no non-cancerous nasopharyngeal specimens showed positive expression of NRP-1. In addition, high NRP-1 expression was significantly associated with advanced clinical stage (P = 0.02), positive recurrence (P = 0.001) and metastasis status (P = 0.001) of NPC. Moreover, the NPC patients with higher NRP-1 expression had shorter overall survival, whereas patients with lower NRP-1 expression had better survival (P < 0.001). Furthermore, the multivariate analysis indicated that the overexpression of NRP-1 protein was an independent prognostic factor for overall survival (P = 0.001) in NPC patients. CONCLUSION: These findings suggest for the first time that NRP-1 upregulation may be a novel biomarker for the prediction of advanced tumor progression and unfavorable prognosis in NPC patients who may benefit from alternative treatment strategy and targeted treatment. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1507827881105018.

Semaphorin 3d signaling defects are associated with anomalous pulmonary venous connections.

Total anomalous pulmonary venous connection (TAPVC) is a potentially lethal congenital disorder that occurs when the pulmonary veins do not connect normally to the left atrium, allowing mixing of pulmonary and systemic blood. In contrast to the extensive knowledge of arterial vascular patterning, little is known about the patterning of veins. Here we show that the secreted guidance molecule semaphorin 3d (Sema3d) is crucial for the normal patterning of pulmonary veins. Prevailing models suggest that TAPVC occurs when the midpharyngeal endothelial strand (MES), the precursor of the common pulmonary vein, does not form at the proper location on the dorsal surface of the embryonic common atrium. However, we found that TAPVC occurs in Sema3d mutant mice despite normal formation of the MES. In these embryos, the maturing pulmonary venous plexus does not anastomose uniquely with the properly formed MES. In the absence of Sema3d, endothelial tubes form in a region that is normally avascular, resulting in aberrant connections. Normally, Sema3d provides a repulsive cue to endothelial cells in this area, establishing a boundary. Sequencing of SEMA3D in individuals with anomalous pulmonary veins identified a phenylalanine-to-leucine substitution that adversely affects SEMA3D function. These results identify Sema3d as a crucial pulmonary venous patterning cue and provide experimental evidence for an alternate developmental model to explain abnormal pulmonary venous connections.

Dysregulation of vascular endothelial growth factors and their neuropilin receptors in the eutopic endometrium of women with endometriosis.

Despite the importance of neuropilins (NRPs) in a number of processes that are altered in endometriosis, such as angiogenesis and neuronal guidance, these molecules have not been previously studied in the disease. Similarly, potent lymphangiogenic factors, vascular endothelial growth factor C (VEGF-C) and VEGF-D, have not been comprehensively investigated in endometriosis. The objective of this study was to examine their expression in women with and without endometriosis. NRPs and VEGFs were quantified in 79 histologically normal uterine tissue samples (37 control and 42 endometriosis, all menstrual cycle phases) using immunohistochemistry and automated cellular imaging analysis. NRP-1 was significantly reduced in women with endometriosis (P = .004). The normal significant menstrual cyclical variations in endometrial NRP-1, NRP-2, and VEGF-C were absent in endometriosis, and VEGF-D was dysregulated. Dysregulated expression of growth factors and receptors, such as NRPs and VEGFs, likely contribute to altered angiogenesis, lymphangiogenesis, neurogenesis and immune function in endometriosis and may reflect altered hormone signals.

The VEGF receptor, neuropilin-1, represents a promising novel target for chronic lymphocytic leukemia patients.

Angiogenesis has been shown to substantially contribute to the progression of chronic lymphocytic leukemia (CLL). Neuropilin-1 (NRP1) represents a receptor for vascular endothelial growth factor (VEGF), which has been reported to be overexpressed in several malignancies. In our study, we characterized mRNA levels of VEGF receptors including NRP1 in a large cohort of CLL patients (n = 114), additionally we performed a detailed characterization of NRP1 expression on B cells, plasmacytoid dendritic cells (PDCs) and regulatory T cells (Tregs). The expression of NRP1 was significantly higher on leukemic lymphocytes compared to control B lymphocytes on mRNA and protein levels (22.72% vs. 0.2%, p = 0.0003, respectively), Tregs (42.6% vs. 16.05%, p = 0.0003) and PDCs (100% vs. 98% p < 0.0001). In functional studies, we found higher NRP1 expression on CLL cells after stimulation with VEGF. The correlation between expression of VEGF receptors: FLT1, NRP1 and FOXP3 expression (r(2) = 0.53, p < 0.0001 and r(2) = 0.49, p < 0.0001, respectively) was observed. Earlier we described the specific Treg reduction during the therapy with thalidomide in vivo. Now we observe the reduction of the NRP1 expression on Tregs in vitro, thereby suggesting a possible target of thalidomide action. In conclusion, NRP1 might represent an interesting link between angiogenesis and tolerance mechanisms and represents interesting target for therapy.

Markers of response for the antiangiogenic agent bevacizumab.

Bevacizumab is the first antiangiogenic therapy proven to slow metastatic disease progression in patients with cancer. Although it has changed clinical practice, some patients do not respond or gradually develop resistance, resulting in rather modest gains in terms of overall survival. A major challenge is to develop robust biomarkers that can guide selection of patients for whom bevacizumab therapy is most beneficial. Here, we discuss recent progress in finding such markers, including the first results from randomized phase III clinical trials evaluating the efficacy of bevacizumab in combination with comprehensive biomarker analyses. In particular, these studies suggest that circulating levels of short vascular endothelial growth factor A (VEGF-A) isoforms, expression of neuropilin-1 and VEGF receptor 1 in tumors or plasma, and genetic variants in VEGFA or its receptors are strong biomarker candidates. The current challenge is to expand this first set of markers and to validate it and implement it into clinical practice. A first prospective biomarker study known as MERiDiAN, which will treat patients stratified for circulating levels of short VEGF-A isoforms with bevacizumab and paclitaxel, is planned and will hopefully provide us with new directions on how to treat patients more efficiently.

Peritumoral brain edema in angiomatous supratentorial meningiomas: an investigation of the vascular endothelial growth factor A pathway.

The aim of this work was to study the vascular endothelial growth factor A (VEGF-A) pathway and peritumoral brain edema (PTBE) through comparison of non-angiomatous and angiomatous meningiomas. Meningiomas are common intracranial tumors, which often have PTBE. VEGF-A is an integral part of PTBE formation and angiogenesis, and the capillary-rich angiomatous meningiomas are known for their PTBE. The VEGF-A receptor VEGFR-2 is responsible for the angiogenic effect of VEGF-A on endothelial cells, which is enhanced by the co-receptor neuropilin-1. Forty non-angiomatous, 22 angiomatous meningiomas, and 10 control tissue samples were collected for the study. Magnetic resonance images were available for 40 non-angiomatous and 10 angiomatous meningiomas. Tissue sections were immunostained for CD34, MIB-1, estrogen- and progesterone receptors. ELISA, chemiluminescence, and RT-qPCR were used for VEGF-A, VEGFR-2, and neuropilin-1 protein and mRNA quantification. Angiomatous meningiomas had larger PTBE (695 vs 218 cm(3) , p = 0.0045) and longer capillary length (3614 vs 605 mm/mm(3) , p < 0.0001). VEGF-A mRNA, neuropilin-1 mRNA, and VEGFR-2 protein levels were higher in angiomatous meningiomas independently of the capillary length (p < 0.05). Neuropilin-1 protein levels were lower in angiomatous meningiomas (p < 0.0001). The VEGF-A pathway and tumor capillary length may be essential for PTBE-formation in meningiomas. Further investigations of this pathway could lead to earlier therapy and targeted pharmacological treatment options.

Decreased semaphorin3A expression correlates with disease activity and histological features of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease of which the pathogenetic mechanisms are not fully understood. Semaphorin3A (Sema3A) has an immune regulatory role. Neuropilin1 (NRP1), the primary receptor for Sema3A, is also a receptor for vascular endothelial growth factor 165 (VEGF 165). It has been shown that Sema3A competitively antagonizes VEGF 165 signaling. This study investigated whether Sema3A is expressed in synovial tissues, and is associated with disease activity and the histological features of synovial tissues from RA patients. METHODS: Human synovial tissues samples were obtained from RA and osteoarthritis (OA) patients. Disease activity of RA patients was calculated using the 28-joint Disease Activity Score based on C-reactive protein (DAS28-CRP). The histological features of RA synovial tissues were evaluated using Rooney's inflammation scoring system. The localization of Sema3A, VEGF 165 and NRP1 positive cells was immunohistochemically determined in synovial tissues. Expression levels of Sema3A, VEGF-A and NRP1 mRNA were determined using quantitative real-time polymerase chain reaction (qPCR). RESULTS: In OA specimens, Sema3A, VEGF 165 and NRP1 proteins were expressed in the synovial lining and inflammatory cells beneath the lining. Immunohistochemistry revealed the protein expression of Sema3A in synovial lining cells was decreased in RA tissues compared with OA samples. qPCR analysis demonstrated a significant reduction of Sema3A mRNA levels in RA synovial tissue samples than in OA and a significant correlation of the ratio of Sema3A/VEGF-A mRNA expression levels with DAS28-CRP (R = -0.449, p = 0.013). Sema3A mRNA levels also correlated with Rooney's inflammation score, especially in perivascular infiltrates of lymphocytes (R = -0.506, p = 0.004), focal aggregates of lymphocytes (R = -0.501, p = 0.005) and diffuse infiltrates of lymphocytes (R = -0.536, p = 0.002). CONCLUSIONS: Reduction of Sema3A expression in RA synovial tissues may contribute to pathogenesis of RA.