Peripheral nerve-derived Sema3A promotes osteogenic differentiation of mesenchymal stem cells through the Wnt/ß-catenin/Nrp1 positive feedback loop.

Sensory nerves play a crucial role in maintaining bone homeostasis by releasing Semaphorin 3A (Sema3A). However, the specific mechanism of Sema3A in regulation of bone marrow mesenchymal stem cells (BMMSCs) during bone remodelling remains unclear. The tibial denervation model was used and the denervated tibia exhibited significantly lower mass as compared to sham operated bones. In vitro, BMMSCs cocultured with dorsal root ganglion cells (DRGs) or stimulated by Sema3A could promote osteogenic differentiation through the Wnt/ß-catenin/Nrp1 positive feedback loop, and the enhancement of osteogenic activity could be inhibited by SM345431 (Sema3A-specific inhibitor). In addition, Sema3A-stimulated BMMSCs or intravenous injection of Sema3A could promote new bone formation in vivo. To sum up, the coregulation of bone remodelling is due to the ageing of BMMSCs and increased osteoclast activity. Furthermore, the sensory neurotransmitter Sema3A promotes osteogenic differentiation of BMMSCs via Wnt/ß-catenin/Nrp1 positive feedback loop, thus promoting osteogenesis in vivo and in vitro.

Neuropilin-1 regulates renin synthesis in juxtaglomerular cells.

Renin is the key enzyme of the systemic renin-angiotensin-aldosterone system, which plays an essential role in regulating blood pressure and maintaining electrolyte and extracellular volume homeostasis. Renin is mainly produced and secreted by specialized juxtaglomerular (JG) cells in the kidney. In the present study, we report for the first time that the conserved transmembrane receptor neuropilin-1 (NRP1) participates in the development of JG cells and plays a key role in renin production. We used the myelin protein zero-Cre (P0-Cre) to abrogate Nrp1 constitutively in P0-Cre lineage-labelled cells of the kidney. We found that the P0-Cre precursor cells differentiate into renin-producing JG cells. We employed a lineage-tracing strategy combined with RNAscope quantification and metabolic studies to reveal a cell-autonomous role for NRP1 in JG cell function. Nrp1-deficient animals displayed abnormal levels of tissue renin expression and failed to adapt properly to a homeostatic challenge to sodium balance. These findings provide new insights into cell fate decisions and cellular plasticity operating in P0-Cre-expressing precursors and identify NRP1 as a novel key regulator of JG cell maturation. KEY POINTS: Renin is a centrepiece of the renin-angiotensin-aldosterone system and is produced by specialized juxtaglomerular cells (JG) of the kidney. Neuropilin-1 (NRP1) is a conserved membrane-bound receptor that regulates vascular and neuronal development, cancer aggressiveness and fibrosis progression. We used conditional mutagenesis and lineage tracing to show that NRP1 is expressed in JG cells where it regulates their function. Cell-specific Nrp1 knockout mice present with renin paucity in JG cells and struggle to adapt to a homeostatic challenge to sodium balance. The results support the versatility of renin-producing cells in the kidney and may open new avenues for therapeutic approaches.

NRP1 Induces Enhanced Stemness and Chemoresistance in Glioma Cells via YAP.

Neuropilin-1 (NRP1), a transmembrane glycoprotein, plays an important role in the malignant progression of gliomas; however, its role in chemoresistance is not fully understood. In this study, we observed the effects of NRP1 on the stemness and chemoresistance of glioma cells and the mediating role of Yes-associated protein (YAP). We constructed NRP1 overexpressing LN-229 glioma cells. Cells were treated with recombinant NRP1 protein (rNRP1) and the YAP inhibitor Super-TDU when necessary. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the sensitivity of cells to temozolomide (TMZ). Sphere and clone formation assays were performed to detect the sphere- and clone-forming abilities of cells. Western blotting was performed to detect cellular CD133, CD44, p-LATS1, and p-YAP protein expression. Immunofluorescence and flow cytometry were used to detect the subcellular localization of YAP and apoptosis, respectively. We found that both NRP1 overexpression and rNRP1 treatment enhanced self-renewal, TMZ resistance, and CD133 and CD44 protein expression in LN-229 cells. NRP1 overexpression and rNRP1 treatment also induced LATS1 and YAP dephosphorylation and YAP nuclear translocation. Super-TDU inhibits NRP1 overexpression-induced enhanced self-renewal and TMZ resistance in LN-229 cells. Our study suggests that NRP1 induces increased stemness in glioma cells, resulting in chemoresistance, and that this effect is associated with YAP activation.

NRP1 interacts with endoglin and VEGFR2 to modulate VEGF signaling and endothelial cell sprouting.

Endothelial cells express neuropilin 1 (NRP1), endoglin (ENG) and vascular endothelial growth factor receptor 2 (VEGFR2), which regulate VEGF-A-mediated vascular development and angiogenesis. However, the link between complex formation among these receptors with VEGF-A-induced signaling and biology is yet unclear. Here, we quantify surface receptor interactions by IgG-mediated immobilization of one receptor, and fluorescence recovery after photobleaching (FRAP) measurements of the mobility of another coexpressed receptor. We observe stable ENG/NRP1, ENG/VEGFR2, and NRP1/VEGFR2 complexes, which are enhanced by VEGF-A. ENG augments NRP1/VEGFR2 interactions, suggesting formation of tripartite complexes bridged by ENG. Effects on signaling are measured in murine embryonic endothelial cells expressing (MEEC+/+) or lacking (MEEC-/-) ENG, along with NRP1 and/or ENG overexpression or knockdown. We find that optimal VEGF-A-mediated phosphorylation of VEGFR2 and Erk1/2 requires ENG and NRP1. ENG or NRP1 increase VEGF-A-induced sprouting, becoming optimal in cells expressing all three receptors, and both processes are inhibited by a MEK1/2 inhibitor. We propose a model where the maximal potency of VEGF-A involves a tripartite complex where ENG bridges VEGFR2 and NRP1, providing an attractive therapeutic target for modulation of VEGF-A signaling and biological responses.

Activation of the aryl hydrocarbon receptor inhibits neuropilin-1 upregulation on IL-2-responding CD4+ T cells.

Neuropilin-1 (Nrp1), a transmembrane protein expressed on CD4+ T cells, is mostly studied in the context of regulatory T cell (Treg) function. More recently, there is increasing evidence that Nrp1 is also highly expressed on activated effector T cells and that increases in these Nrp1-expressing CD4+ T cells correspond with immunopathology across several T cell-dependent disease models. Thus, Nrp1 may be implicated in the identification and function of immunopathologic T cells. Nrp1 downregulation in CD4+ T cells is one of the strongest transcriptional changes in response to immunoregulatory compounds that act though the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. To better understand the link between AhR and Nrp1 expression on CD4+ T cells, Nrp1 expression was assessed in vivo and in vitro following AhR ligand treatment. In the current study, we identified that the percentage of Nrp1 expressing CD4+ T cells increases over the course of activation and proliferation in vivo. The actively dividing Nrp1+Foxp3- cells express the classic effector phenotype of CD44hiCD45RBlo, and the increase in Nrp1+Foxp3- cells is prevented by AhR activation. In contrast, Nrp1 expression is not modulated by AhR activation in non-proliferating CD4+ T cells. The downregulation of Nrp1 on CD4+ T cells was recapitulated in vitro in cells isolated from C57BL/6 and NOD (non-obese diabetic) mice. CD4+Foxp3- cells expressing CD25, stimulated with IL-2, or differentiated into Th1 cells, were particularly sensitive to AhR-mediated inhibition of Nrp1 upregulation. IL-2 was necessary for AhR-dependent downregulation of Nrp1 expression both in vitro and in vivo. Collectively, the data demonstrate that Nrp1 is a CD4+ T cell activation marker and that regulation of Nrp1 could be a previously undescribed mechanism by which AhR ligands modulate effector CD4+ T cell responses.

Susceptibility of bovine to SARS-CoV-2 variants of concern: insights from ACE2, AXL, and NRP1 receptors.

The possibilities of cross-species transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) between humans and important livestock species are not yet known. Herein, we used the structural and genetic alignment and surface potential analysis of the amino acid (aa) in angiotensin-converting enzyme 2 (ACE2), tyrosine kinase receptor UFO (AXL), and neuropilin 1 (NRP1) in different species with substantial public health importance. The residues interfacing with the N-terminal domain (NTD) or receptor-binding domain (RBD) of S were aligned to screen the critical aa sites that determined the susceptibility of the SARS-CoV-2 to the host. We found that AXL and NRP1 proteins might be used as the receptors of SARS-CoV-2 in bovines. However, ACE2 protein may not be considered to be involved in the cross-species transmission of SARS-CoV-2 VOCs in cattle because the key residues of the ACE2-S-binding interface were different from those in known susceptible species. This study indicated that emerging SARS-CoV-2 variants potentially expand species tropism to bovines through AXL and NRP1 proteins.

VEGF/Nrp1/HIF-1α promotes proliferation of hepatocellular carcinoma through a positive feedback loop.

To investigate the role of neuropilin1 (Nrp1) in glucose metabolism and proliferation of hepatocellular carcinoma (HCC) cells and to analyze its mechanism of action. The CRISPR gene knockout technique was used to knock out the Nrp1 gene in two HCC cell lines. The effect of Nrp1 on the proliferation of HCC cells was assessed in the CCK8 assay and plate cloning assay. The expression levels of glucose consumption, lactate production, and essential proteins of the glycolytic pathway were detected to explore the effect of Nrp1 on glucose metabolism in HCC cells. Using CoCl2 to revert the expression of hypoxia inducible factor-1α (HIF-1α), the role of HIF-1α in the pro-HCC cell metabolism of Nrp1 were demonstrated. The protein synthesis inhibitor CHX and proteasome inhibitor MG-132 was used to analyze the molecular mechanism of action of Nrp1 on HIF-1α. The Kaplan-Meier method was used to calculate survival rates and plot survival curves. Based on the CCK8 assay and plate cloning assay, we found that Nrp1 knockout significantly inhibited the proliferation of HCC cells. Nrp1 inhibitor suppressed lactate production and glucose consumption in HCC cells. Knockout of Nrp1 decreased the expression of glycolytic pathway-related proteins and HIF-1α protein. Furthermore, by joint use of CoCl2 and NRP1 knockout, we confirmed that reverting HIF-1α expression could reverse the effect of Nrp1 knockout on HCC cell metabolism in vitro. Mechanistically, Nrp1 showed a close correlation with the stability of HIF-1α protein in protein stability assay. Finally, we revealed that high expression of Nrp1 in HCC tissues was associated with poor overall survival and disease-free survival of the patients. Nrp1 accelerates glycolysis and promotes proliferation of HCC by regulating HIF-1α protein stability and through the VEGF/Nrp1/HIF-1α positive feedback loop.

Long noncoding RNA PVT1 predicts poor prognosis and promotes the progression of colorectal cancer through the miR-24-3p/NRP1 axis in zebrafish xenografts.

Long noncoding RNAs (lncRNAs) play important roles in the progression of human cancer. It is reported that lncRNA plasmacytoma variant translocation 1 (PVT1) is involved in colorectal cancer (CRC), however, the underlying mechanism remains to be explored deeply, especially by in vivo models. In the present study, bioinformatics analysis showed that the expression level of PVT1 was upregulated in CRC tissues and highly associated with poor prognosis of CRC patients. In cultured CRC cells, knockdown of PVT1 inhibited cell proliferation and migration of CRC cells, while overexpression of PVT1 promoted the progression of CRC cells. In zebrafish xenografts, the silencing of PVT1 also suppressed the growth and metastasis of CRC cells. For mechanism studies, the binding relationships among PVT1, miR-24-3p, and Neuropilin 1 (NRP1) were predicted by starBase firstly. The luciferase reporter assays verified that PVT1 and NRP1 could bind with miR-24-3p directly. Further studies showed miR-24-3p negatively regulated the progression of CRC cells, the inhibition of miR-24-3p counteracted the repression effects of CRC progression when knocking down PVT1. In addition, the expression of NRP1 was regulated by PVT1, and NRP1 overexpression could partially rescue the inhibition effects of CRC progression when knocking down PVT1 in vitro and in vivo. Our study reveals that PVT1 promotes the proliferation and metastasis of CRC via regulating the miR-24-3p/NRP1 axis, which provides a prognosis biomarker and a potential therapeutic target for CRC patients.

The semaphorin 3A/neuropilin-1 pathway promotes clonogenic growth of glioblastoma via activation of TGF-ß signaling.

Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence; thus, understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the CNS, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF-ß receptor 1 (TGF-ßR1), which in turn leads to activation of canonical TGF-ß signaling in both GSCs and NPCs. TGF-ß signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A/NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF-ß pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A/NRP1/TGF-ßR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.

Neuropilin 1 promotes unilateral ureteral obstruction-induced renal fibrosis via RACK1 in renal tubular epithelial cells.

Neuropilin 1 (NRP1) is a single-channel transmembrane glycoprotein whose role and mechanism in renal fibrosis remain incompletely elucidated. Therefore, we investigated the effect of NRP1 on renal fibrosis and its potential mechanism. NRP1 expression in the renal sections from patients with chronic kidney disease (CKD) and a unilateral ureteral obstruction (UUO) mouse model was detected. Nrp1 overexpression or knockdown plasmid was transfected into mice, TKPTS mouse kidney proximal tubular epithelial cells (TECs), and rat kidney fibroblasts, after which pathological injury evaluation and fibrosis marker detection were conducted. The direct interaction of the receptor of activated protein C kinase 1 (RACK1) with NRP1 was validated by immunoprecipitation and Western blot analysis. We found that the upregulated renal NRP1 expression in patients with CKD was located in proximal TECs, consistent with the degree of interstitial fibrosis. In the UUO mouse model, NRP1 expression was upregulated in the kidney, and overexpression of Nrp1 increased the mRNA and protein expression of fibronectin (Fn) and α-smooth muscle actin (α-SMA), whereas Nrp1 knockdown significantly reduced Fn and α-SMA expression and downregulated the inflammatory response. NRP1 promoted transforming growth factor ß1 (TGF-ß1)-induced profibrotic responses in the TKPTS cells and fibroblasts, and Nrp1 knockdown partially reversed these responses. Immunoprecipitation combined with liquid chromatography-tandem mass spectrometry verified that NRP1 can directly bind to RACK1, and Rack1 knockdown reversed the NRP1-induced fibrotic response. In summary, NRP1 may enhance the TGF-ß1 pathway by binding to RACK1, thus promoting renal fibrosis.NEW & NOTEWORTHY Although a few studies have confirmed the correlation between neuropilin 1 (NRP1) and renal diseases, the mechanism of NRP1 in renal fibrosis remains unclear. Here, we investigated the effects of NRP1 on renal fibrosis through in vitro and in vivo experiments and explored the possible downstream mechanisms. We found that NRP1 can stimulate the TGF-ß1 signaling pathway, possibly by binding to RACK1, thereby promoting renal fibrosis.

Emerging SARS-CoV-2 variants of concern potentially expand host range to chickens: insights from AXL, NRP1 and ACE2 receptors.

BACKGROUND: The possibilities of cross-species transmission of SARS-CoV-2 variants of concern (VOCs) between humans and poultry species are unknown. The analysis of the structure of receptor was used to investigate the potential of emerging SARS-CoV-2 VOCs to expand species tropism to chickens based on the interaction between Spike (S) protein and tyrosine kinase receptor UFO (AXL), angiotensin-converting enzyme 2 (ACE2), and neuropilin 1 (NRP1) with substantial public health importance. METHODS: The structural and genetic alignment and surface potential analysis of the amino acid (aa) in ACE2, AXL, and NRP1 in human, hamster, mouse, mink, ferret, rhesus monkey and chickens were performed by Swiss-Model and pymol software. The critical aa sites that determined the susceptibility of the SARS-CoV-2 to the host were screened by aligning the residues interfacing with the N-terminal domain (NTD) or receptor-binding domain (RBD) of Spike protein. RESULTS: The binding modes of chickens AXL and ACE2 to S protein are similar to that of the ferret. The spatial structure and electrostatic surface potential of NRP1 showed that SARS-CoV-2 VOCs could not invade chickens through NRP1 easily. CONCLUSION: These results suggested that emerging SARS-CoV-2 VOCs potentially expand the host range to chickens mainly through ACE2 and AXL receptors, while NRP1 receptor may rarely participate in the future epidemic of coronavirus disease 2019 in chickens.

Conditional, Tissue-Specific CRISPR/Cas9 Vector System in Zebrafish Reveals the Role of Nrp1b in Heart Regeneration.

BACKGROUND: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) technology-mediated genome editing has significantly improved the targeted inactivation of genes in vitro and in vivo in many organisms. Neuropilins play crucial roles in zebrafish heart regeneration, heart failure in mice, and electrical remodeling after myocardial infarction in rats. But the cell-specific functions of nrp1 have not been described before. In this study, we have investigated the role of nrp1 isoforms, including nrp1a and nrp1b, in cardiomyocytes during cardiac injury and regeneration in adult zebrafish hearts. METHODS: In this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specific cmlc2 promoter drives Cas9 expression to silence the nrp1 gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes a unique tool to regulate the gene knockout in both the developmental and adult stages and hence widens the possibility of loss-of-function studies in zebrafish at different stages of development and adulthood. Using this approach, we investigated the role of neuropilin isoforms nrp1a and nrp1b in response to cardiac injury and regeneration in adult zebrafish hearts. RESULTS: We observed that both the isoforms (nrp1a and nrp1b) are upregulated after the cryoinjury. Interestingly, the nrp1b knockout significantly delayed heart regeneration and impaired cardiac function in the adult zebrafish after cryoinjury, demonstrated by reduced heart rate, ejection fractions, and fractional shortening. In addition, we show that the knockdown of nrp1b but not nrp1a induces activation of the cardiac remodeling genes in response to cryoinjury. CONCLUSIONS: To our knowledge, this study is novel where we have reported a heat-shock-mediated conditional knockdown of nrp1a and nrp1b isoforms using CRISPR/Cas9 technology in the cardiomyocyte in zebrafish and furthermore have identified a crucial role for the nrp1b isoform in zebrafish cardiac remodeling and eventually heart function in response to injury.

New Crystal Form of Human Neuropilin-1 b1 Fragment with Six Electrostatic Mutations Complexed with KDKPPR Peptide Ligand.

Neuropilin 1 (NRP1), a cell-surface co-receptor of a number of growth factors and other signaling molecules, has long been the focus of attention due to its association with the development and the progression of several types of cancer. For example, the KDKPPR peptide has recently been combined with a photosensitizer and a contrast agent to bind NRP1 for the detection and treatment by photodynamic therapy of glioblastoma, an aggressive brain cancer. The main therapeutic target is a pocket of the fragment b1 of NRP1 (NRP1-b1), in which vascular endothelial growth factors (VEGFs) bind. In the crystal packing of native human NRP1-b1, the VEGF-binding site is obstructed by a crystallographic symmetry neighbor protein, which prevents the binding of ligands. Six charged amino acids located at the protein surface were mutated to allow the protein to form a new crystal packing. The structure of the mutated fragment b1 complexed with the KDKPPR peptide was determined by X-ray crystallography. The variant crystallized in a new crystal form with the VEGF-binding cleft exposed to the solvent and, as expected, filled by the C-terminal moiety of the peptide. The atomic interactions were analyzed using new approaches based on a multipolar electron density model. Among other things, these methods indicated the role played by Asp320 and Glu348 in the electrostatic steering of the ligand in its binding site. Molecular dynamics simulations were carried out to further analyze the peptide binding and motion of the wild-type and mutant proteins. The simulations revealed that specific loops interacting with the peptide exhibited mobility in both the unbound and bound forms.

Systematic pan-cancer analysis identified neuropilin 1 as an immunological and prognostic biomarker.

Neuropilin 1 (NRP1) is a transmembrane glycoprotein, nontyrosine kinase receptor that plays an important role in axonal growth and angiogenesis in the nervous system. Although currently more and more studies have shown that NRP1 plays an important role in some cancers, no systematic pan-cancer analysis of NRP-1 has been performed to date. Therefore, we aimed to investigate the associated immune function and prognostic value of NRP1 in 33 tumors of various cancer types. In this study, based on The Cancer Genome Atlas, Cancer Cell Line Encyclopedia, Genotype Tissue Expression, cBioportal for cancer genomics, and Human Protein Atlas (HPA databases), various bioinformatics analysis methods were used to investigate the potential carcinogenic effects of NRP1 activation, pan-cancer analysis of NRP1 expression, and the relationship between NRP1 expression and prognosis indicators including overall survival, disease-specific survival, disease-free interval, and progression-free interval, tumor mutational burden (TMB), and microsatellite instability (MSI). The results showed that NRP1 was highly expressed in most tumors. In addition, NRP1 was found to be positively or negatively correlated with the prognosis of different tumors. Also, the expression of NRP1 was associated with TMB and MSI in in 27 and 21 different types of tumors, respectively, and with DNA methylation in almost all the various types of tumors. The expression of the NRP1 gene was negatively correlated with the infiltration levels of most immune cells. In addition, the correlation between the level of immune cell infiltration and NRP1 expression varied according to immune cell subtype. Our study suggests that NRP1 plays an important role in tumor development and tumor immunity and could potentially be used as a prognostic indicator in a variety of malignancies.

Paradoxical expression of NRP1 in decidual stromal and immune cells reveals a novel inflammation balancing mechanism during early pregnancy.

OBJECTIVE AND DESIGN: To investigate the balancing mechanisms between decidualization-associated inflammation and pregnancy-related immunotolerance. MATERIAL OR SUBJECTS: Decidual samples from women with normal pregnancy (n = 58) or unexplained spontaneous miscarriage (n = 13), peripheral blood from normal pregnancy and endometria from non-pregnancy (n = 10) were collected. Primary endometrial stromal cells (ESCs), decidual stromal cells (DSCs), decidual immune cells (DICs) and peripheral blood mononuclear cells (PBMCs) were isolated. TREATMENT: The plasmid carrying neuropilin-1 (NRP1) gene was transfected into ESC for overexpression. To induce decidualization in vitro, ESCs were treated with a combination of 10 nM estradiol, 100 nM progesterone and 0.5 mM cAMP. Anti-Sema3a and anti-NRP1 neutralizing antibodies were applied to block the ligand-receptor interactions. METHODS: RNA-seq analysis was performed to identify differentially expressed genes in DSCs and DICs, and NRP1 expression was verified by Western blotting and flow cytometry. The secretion of inflammatory mediators was measured using a multifactor cytometric bead array. The effects of Sema3a-NRP1 pathway on DICs were determined by flow cytometry. Statistical differences between groups were compared using the T test and one way or two-way ANOVA. RESULTS: Combined with five RNA-seq datasets, NRP1 was the only immune checkpoint changing oppositely between DSCs and DICs. The decreased expression of NRP1 in DSCs allowed intrinsic inflammatory responses required for decidualization, while its increased expression in DICs enhanced tolerant phenotypes beneficial to pregnancy maintenance. DSC-secreted Sema3a promoted immunosuppression in DICs via NRP1 binding. In women with miscarriage, NRP1 was abnormally elevated in DSCs but diminished in decidual macrophages and NK cells. CONCLUSION: NRP1 is a multifunctional controller that balances the inflammatory states of DSCs and DICs in gravid uterus. Abnormal expression of NRP1 is implicated in miscarriage.

Neuropilin-1 is essential for vascular endothelial growth factor A-mediated increase of sensory neuron activity and development of pain-like behaviors.

ABSTRACT: Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that binds numerous ligands including vascular endothelial growth factor A (VEGFA). Binding of this ligand to NRP-1 and the co-receptor, the tyrosine kinase receptor VEGFR2, elicits nociceptor sensitization resulting in pain through the enhancement of the activity of voltage-gated sodium and calcium channels. We previously reported that blocking the interaction between VEGFA and NRP-1 with the Spike protein of SARS-CoV-2 attenuates VEGFA-induced dorsal root ganglion (DRG) neuronal excitability and alleviates neuropathic pain, pointing to the VEGFA/NRP-1 signaling as a novel therapeutic target of pain. Here, we investigated whether peripheral sensory neurons and spinal cord hyperexcitability and pain behaviors were affected by the loss of NRP-1. Nrp-1 is expressed in both peptidergic and nonpeptidergic sensory neurons. A CRIPSR/Cas9 strategy targeting the second exon of nrp-1 gene was used to knockdown NRP-1. Neuropilin-1 editing in DRG neurons reduced VEGFA-mediated increases in CaV2.2 currents and sodium currents through NaV1.7. Neuropilin-1 editing had no impact on voltage-gated potassium channels. Following in vivo editing of NRP-1, lumbar dorsal horn slices showed a decrease in the frequency of VEGFA-mediated increases in spontaneous excitatory postsynaptic currents. Finally, intrathecal injection of a lentivirus packaged with an NRP-1 guide RNA and Cas9 enzyme prevented spinal nerve injury-induced mechanical allodynia and thermal hyperalgesia in both male and female rats. Collectively, our findings highlight a key role of NRP-1 in modulating pain pathways in the sensory nervous system.

Neuropilin-1 Knockout and Rescue Confirms Its Role to Promote Metastasis in MDA-MB-231 Breast Cancer Cells.

Breast cancer (BC) metastasis remains a leading cause of female mortality. Neuropilin-1 (NRP-1) is a glycoprotein receptor that plays ligand-dependent roles in BC. Clinical studies indicate its correlation with metastatic disease; however, its functional role in BC metastasis remains uncertain. CRISPR-Cas9 was used to knockout the NRP-1 gene in MDA-MB-231 BC cells, and the effects on metastasis were determined using an orthotopic mouse engraftment model. NRP-1 expression in knockout cells was rescued using a recombinant cDNA with a silent mutation in the sgRNA target-adjacent PAM sequence. Differentially expressed genes between NRP-1 knockout and control cells were determined using whole-transcriptome sequencing and validated using real-time PCR. NRP-1KO cells showed a pronounced reduction in the metastasis to the lungs. KEGG pathway analysis of the transcriptome data revealed that PI3K and ECM receptor interactions were among the top altered pathways in the NRP-1KO cells. In addition, reduction in metastasis enhancers proteins, Integrin-ß3 and Tenascin-C, and genes CCL20 and FN1 and upregulation of metastasis suppressor genes, ACVRL and GPX3 in NRP-1KO were detected. These findings provide evidence for a functional role for NRP-1 in BC metastasis, supporting further exploration of NRP-1 and the identified genes as targets in treating metastatic BC.

Exposure to Valproic acid (VPA) resulted in alterations in the expression of angiogenic genes (NRP-1, VEGFA, VEGFR-2 and sFlt1) and histological modifications in the placenta of mice (Mus musculus).

Valproic acid (VPA), an anti-epileptic drug (AED), has been reported to exhibit anti-angiogenic properties. This study aimed to examine the impact of VPA on the expression of NRP-1 and additional angiogenic factors, as well as angiogenesis, in mouse placenta. Pregnant mice were divided into four groups: control (K), solvent control (KP), VPA treatment at a dose of 400 mg/kg body weight (BW) (P1), and VPA treatment at a dose of 600 mg/kg BW (P2). The mice were subjected to daily treatment via gavage from embryonic day (E) 9 to E14 and E9 to E16. Histological analysis was performed to evaluate Microvascular Density (MVD) and percentage of the placental labyrinth area. In addition, a comparative analysis of Neuropilin-1 (NRP-1), vascular endothelial growth factor (VEGFA), vascular endothelial growth factor receptor (VEGFR-2), and soluble (sFlt1) expression was conducted in relation to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results of the MVD analysis and percentage of labyrinth area in the E14 and E16 placentas indicated that the treated groups were significantly lower than the control group. The relative expression levels of NRP-1, VEGFA, and VEGFR-2 in the treated groups were lower than those in the control group at E14 and E16. Meanwhile, the relative expression of sFlt1 in the treated groups at E16 was significantly higher than in the control group. Changes in the relative expression of these genes inhibit angiogenesis regulation in the mouse placenta, as evidenced by reduced MVD and a smaller percentage of the labyrinth area.

ACE-2, TMPRSS2, and Neuropilin-1 Receptor Expression on Human Brain Astrocytes and Pericytes and SARS-CoV-2 Infection Kinetics.

Angiotensin Converting Enzyme 2 (ACE-2), Transmembrane Serine Protease 2 (TMPRSS-2) and Neuropilin-1 cellular receptors support the entry of SARS-CoV-2 into susceptible human target cells and are characterized at the molecular level. Some evidence on the expression of entry receptors at mRNA and protein levels in brain cells is available, but co-expression of these receptors and confirmatory evidence on brain cells is lacking. SARS-CoV-2 infects some brain cell types, but infection susceptibility, multiple entry receptor density, and infection kinetics are rarely reported in specific brain cell types. Highly sensitive Taqman ddPCR, flow-cytometry and immunocytochemistry assays were used to quantitate the expression of ACE-2, TMPRSS-2 and Neuropilin-1 at mRNA and protein levels on human brain-extracted pericytes and astrocytes, which are an integral part of the Blood-Brain-Barrier (BBB). Astrocytes showed moderate ACE-2 (15.9 ± 1.3%, Mean ± SD, n = 2) and TMPRSS-2 (17.6%) positive cells, and in contrast show high Neuropilin-1 (56.4 ± 39.8%, n = 4) protein expression. Whereas pericytes showed variable ACE-2 (23.1 ± 20.7%, n = 2), Neuropilin-1 (30.3 ± 7.5%, n = 4) protein expression and higher TMPRSS-2 mRNA (667.2 ± 232.3, n = 3) expression. Co-expression of multiple entry receptors on astrocytes and pericytes allows entry of SARS-CoV-2 and progression of infection. Astrocytes showed roughly four-fold more virus in culture supernatants than pericytes. SARS-CoV-2 cellular entry receptor expression and "in vitro" viral kinetics in astrocytes and pericytes may improve our understanding of viral infection "in vivo". In addition, this study may facilitate the development of novel strategies to counter the effects of SARS-CoV-2 and inhibit viral infection in brain tissues to prevent the spread and interference in neuronal functions.

Sodium Valproate Enhances Semaphorin 3A-mediated Anti-angiogenesis and Tumor Growth Inhibition in Human Osteosarcoma Cells.

BACKGROUND/AIM: Class 3 semaphorins, including semaphorin 3A (SEMA3A), are known endogenous angiogenesis inhibitors associated with endothelial cell migration and proliferation, and have been identified in many cancer cells. SEMA3A suppresses tumor angiogenesis by competing with VEGF, but tumors are known to have active angiogenesis, suggesting that expression of SEMA3A and its receptors is epigenetically restrained. To overcome this condition, we aimed to use histone deacetylase (HDAC) inhibitors to enhance the SEMA3A expression in osteosarcoma (OS) cells, thereby suppressing angiogenesis and inhibiting their proliferation and metastasis. MATERIALS AND METHODS: OS cell lines and human microvascular endothelial (HMVE) cells were treated with HDAC inhibitors such as sodium valproate (VPA) and Trichostatin A (TSA). Changes in the SEMA3A expression and its related receptors at the mRNA and protein levels, as well as the inhibitory effects on tumor angiogenesis, were investigated. RESULTS: VPA and TSA increased the expression of SEMA3A and its receptor NRP1, without inducing PLXNA1 in OS cells. Similarly, SEMA3A and NRP1 expression was increased in HMVE cells, but no growth inhibition was observed. Furthermore, SEMA3A induced by VPA in OS cell culture medium inhibited vascular tube formation of HMVE cells, and overexpression of SEMA3A enhanced OS cell growth inhibition. This growth-inhibitory effect of SEMA3A induced G1/S cell cycle arrest in OS cells. CONCLUSION: HDAC inhibitors have anti-angiogenic and anti-tumor activities that may be, in part, mediated via the SEMA3A/NRP1/PLXNA1 autocrine and paracrine pathways.