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Clearance of airway intruders by immune cells is required to resolve infectious pneumonia. However, the molecular mechanisms underlying this process remain elusive. Here, we demonstrated that alveolar macrophage (AM)-derived neuropilin 2 (NRP2) plays an essential role in controlling severe pneumonia by enhancing microbial clearance. Mice with conditional deletion of the NRP2 gene in AM had persistent bacteria, uncontrolled neutrophil influx, and decreased survival during Escherichia coli-induced pneumonia. In vitro assays demonstrated that NRP2 could bind to CD11b + Ly6G lo/+ neutrophils and promote their capacities in phagocytosis and killing of bacteria, which is partially contributed to the increased expression of TLR4 and TNF-a. These findings collectively revealed that AM-derived NRP2 protects the lungs from unwanted injury by promoting the clearance of invading pathogens. This study might provide a promising diagnostic biomarker and therapeutic target for severe pneumonia., (©2022 Society for Leukocyte Biology.)

Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secreted protein, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle injury; however, its physiological significance is still unknown. Here we show that Sema3A impacts slow-twitch fiber generation through a signaling pathway, cell-membrane receptor (neuropilin2-plexinA3) → myogenin-myocyte enhancer factor 2D → slow myosin heavy chain. This novel axis was found by small interfering RNA-transfection experiments in myoblast cultures, which also revealed an additional element that Sema3A-neuropilin1/plexinA1, A2 may enhance slow-fiber formation by activating signals that inhibit fast-myosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreER T2 -Sema3A fl ° x activated by tamoxifen-i.p. injection) provided direct in vivo evidence for the Sema3A-driven program, by showing that slow-fiber generation and muscle endurance were diminished after repair from cardiotoxin-injury of gastrocnemius muscle. Overall, the findings highlight an active role for satellite cell-secreted Sema3A ligand as a key "commitment factor" for the slow-fiber population during muscle regeneration. Results extend our understanding of the myogenic stem-cell strategy that regulates fiber-type differentiation and is responsible for skeletal muscle contractility, energy metabolism, fatigue resistance, and its susceptibility to aging and disease. Stem Cells 2017;35:1815-1834., (© 2017 AlphaMed Press.)

Class 3 semaphorins (Semas) are soluble proteins that are well recognized for their role in guiding axonal migration during neuronal development. In the immune system, Sema3A has been shown to influence murine dendritic cell (DC) migration by signaling through a neuropilin (NRP)-1/plexin-A1 coreceptor axis. Potential roles for class 3 Semas in human DCs have yet to be described. We tested the hypothesis that Sema3A, -3C, and -3F, each with a unique NRP-1 and/or NRP-2 binding specificity, influence human DC migration. In this report, we find that although NRP-1 and NRP-2 are expressed in human immature DCs (imDCs), NRP-2 expression increases as cells mature further, whereas expression of NRP-1 declines dramatically. Elevated levels of RNA encoding plexin-A1 and -A3 are present in both imDCs and mature DC (mDCs), supporting the relevance of Sema/NRP/plexin signaling pathways in these cells. Sema3A, -3C, and -3F bind to human DCs, with Sema3F binding predominantly through NRP-2. The binding of these Semas leads to reorganization of actin filaments at the plasma membrane and increased transwell migration in the absence or presence of chemokine CCL19. Microfluidic chamber assays failed to demonstrate consistent changes in speed of Sema3C-treated DCs, suggesting increased cell deformability as a possible explanation for enhanced transwell migration. Although monocytes express RNA encoding Sema3A, -3C, and -3F, only RNA encoding Sema3C increases robustly during DC differentiation. These data suggest that Sema3A, -3C, and -3F, likely with coreceptors NRP-1, NRP-2, and plexin-A1 and/or -A3, promote migration and possibly other activities of human DCs during innate and adaptive immune responses., (© Society for Leukocyte Biology.)

Background: A major hallmark of malignant progression in human astrocytomas is the formation of new blood vessels. Antiangiogenic therapy using the anti-vascular endothelial growth factor (VEGF)-antibody bevacizumab leads to increased progression-free survival in glioblastoma patients but does not influence their overall survival. To date, it is unclear why antiangiogenic therapy fails in many glioblastoma patients, while a small subpopulation profits considerably from this treatment., Methods: The aim of our study was to determine the expression of VEGF-A and its (co-) receptors by immunohistochemistry and to test the association with patient survival in 350 glioma patients. Additionally, VEGF-A expression was analyzed by in-situ hybridization. In 18 patients, the protein expression was compared with the bevacizumab response according to extended and modified RANO criteria., Results: We found a heterogeneous expression pattern of VEGF and its receptors in glioblastoma patients with significantly lower levels in WHO grade II and III tumors and normal-appearing brain tissue (P < .001). Pilocytic astrocytomas (WHO grade I) showed significantly higher VEGFR-1, -2 and neuropilin-1 levels as compared to WHO grade II and III astrocytomas (P < .01) but at lower levels than glioblastomas. The expression of neuropilin-2 was low in all tumors. There was neither a significant correlation between protein expression and patient survival nor between protein levels and bevacizumab response after modified RANO criteria., Conclusion: Since our data indicate that beneficial response to bevacizumab treatment is independent of the expression of VEGF-A and its (co-) receptors, further investigation is needed to decipher the underlying mechanisms of antiangiogenic treatment response., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Neuropilin-2 (Nrp2) is a coreceptor for vascular endothelial growth factor-D (VEGF-D) that is expressed on the surface of endothelial cells. Recently, Nrp2 was shown to play a role in lymph node metastasis and promotion of cancer cell migration. VEGF-D also promotes lymphangiogenesis, which in turn promotes tumor metastasis., Objective: The aim was to study the role of neuropilin-2 in lymph node metastasis in human papillary thyroid carcinoma (PTC)., Design: Expression of Nrp2 was studied by immunohistochemistry and the relationship between Nrp2 expression and lymph node metastasis, VEGF-D expression and other established clinicopathological variables were analyzed in PTC. The effects of neutralizing anti-Nrp2 antibody on VEGF-D-induced invasion and migration were assessed in PTC cell lines., Results: Nrp2 expression was observed in 64.3% (36 of 56) of the PTC patients. Nrp2 expression was significantly correlated with lymph node metastasis (P = 0.0216) and VEGF-D expression (P = 0.0034). VEGF-D was shown to promote filopodia formation and cancer cell migration and invasion by K1 and B-CPAP cells. These responses were significantly blocked by neutralizing anti-Nrp2 antibody., Conclusion: Nrp2 expression was correlated with lymph node metastasis and VEGF-D expression in PTC. Our data also showed a role for Nrp2 in regulating VEGF-D-induced invasion and migration in vitro.

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.

Although much is known about the biology of vascular endothelial growth factor (VEGF) and its cognate receptors (VEGFRs), VEGFR1 and VEGFR2, little is known about the roles of the VEGFRs neuropilin (NP)-1 and NP-2 in the primate endometrium. In this study, we investigated the cellular localization and hormonal regulation of NP-1 and NP-2 mRNA by in situ hybridization in the endometrium of ovariectomized, hormonally cycled rhesus macaques and women during the natural menstrual cycle. NP-1 mRNA was highly expressed in vascular endothelium and in stromal cells, but in these cells, NP-1 expression did not change during the menstrual cycle. However, NP-1 mRNA was also expressed in the luminal epithelium (not the glands), and its expression in these cells was elevated during the mid- to late proliferative phase and completely suppressed during the secretory phase. The increase in NP-1 level in the luminal epithelium was estradiol dependent because such expression was not detectable in the absence of estradiol in ovariectomized, hormone-deprived animals. Moreover, NP-1 expression in the luminal epithelium was highly correlated with the degree of proliferation in these cells. A recent study showed that blockade of VEGF action can inhibit luminal epithelial cell proliferation, but there is no evidence of VEGFR1 and VEGFR2 expression in these cells. Therefore, NP-1 may be the relevant VEGFR that mediates proliferation in this epithelium. NP-2 mRNA, unlike NP-1, was expressed only by the endothelium of veins, and in these cells, its expression was hormonally regulated in the converse manner: it was very low during the proliferative phase and high during the secretory phase. The increased permeability and edema observed during the secretory phase in the primate endometrium may be mediated in part by VEGF-NP-2 interaction. In the human endometrium, the pattern of expression and cellular localization of both NP-1 and NP-2 during the menstrual cycle were essentially identical with that seen in the rhesus macaque endometrium. These are the first data to specify the hormonal regulation and cell-specific expression of NP-1 and NP-2 mRNA in the endometrium of both women and nonhuman primates. The findings extend our understanding of VEGF action in the primate endometrium.

Vascular endothelial growth factor (VEGF) is a potent regulator of placental vascular function. Endothelial dysfunction is a key factor associated with preeclampsia. In this study, we examined expression of VEGF, endocrine gland-derived VEGF (EG-VEGF), VEGF receptors 1 and 2 (VEGFR-1 and VEGFR-2), and neuropilin-1 and -2 (NP-1 and NP-2) in human placentas from women with normal and preeclamptic (PE) pregnancies using quantitative or semiquantitative PCR. We found that total VEGF mRNA expression was increased 2.8-fold (P < 0.05), along with increases in mRNA expression of VEGF121, 165, and 189 (P < 0.05; 1.7-, 1.9-, and 1.8-fold, respectively) in PE vs. normal placentas. Expression of VEGFR-1 mRNA, but not EG-VEGF and the other three VEGF receptors studied, was elevated (P < 0.05) 2.7-fold in PE vs. normal placentas. Protein expression of VEGF and its four receptors was determined using Western blot analysis. For VEGF, two major isoforms (VEGF165 and 189) were detected. For VEGFR-1, VEGFR-2, NP-1, and NP-2, one major band was observed at 180, 235, 130, and 130 kDa, respectively. All of these bands were corresponding to their positive controls. Of these five proteins studied, only VEGFR-1 levels were increased (P < 0.05; 1.7-fold) in PE placentas. The expression of VEGF and the four VEGF receptors was confirmed using immunohistochemistry. They were primarily present in syncytiotrophoblasts and endothelial cells of villous capillaries and large vessels. Thus, together with previous reports that VEGFR-1 mediates trophoblast function and inhibits VEGF-induced angiogenesis and endothelium-dependent vasodilation, these data suggest that the increased VEGFR-1 expression may alter VEGF- mediated function on trophoblast and endothelial cells in PE placentas.