Receptors Involved in COVID-19-Related Anosmia: An Update on the Pathophysiology and the Mechanistic Aspects.

Olfactory perception is an important physiological function for human well-being and health. Loss of olfaction, or anosmia, caused by viral infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has received considerable attention, especially in persistent cases that take a long time to recover. This review discusses the integration of different components of the olfactory epithelium to serve as a structural and functional unit and explores how they are affected during viral infections, leading to the development of olfactory dysfunction. The review mainly focused on the role of receptors mediating the disruption of olfactory signal transduction pathways such as angiotensin converting enzyme 2 (ACE2), transmembrane protease serine type 2 (TMPRSS2), neuropilin 1 (NRP1), basigin (CD147), olfactory, transient receptor potential vanilloid 1 (TRPV1), purinergic, and interferon gamma receptors. Furthermore, the compromised function of the epithelial sodium channel (ENaC) induced by SARS-CoV-2 infection and its contribution to olfactory dysfunction are also discussed. Collectively, this review provides fundamental information about the many types of receptors that may modulate olfaction and participate in olfactory dysfunction. It will help to understand the underlying pathophysiology of virus-induced anosmia, which may help in finding and designing effective therapies targeting molecules involved in viral invasion and olfaction. To the best of our knowledge, this is the only review that covered all the receptors potentially involved in, or mediating, the disruption of olfactory signal transduction pathways during COVID-19 infection. This wide and complex spectrum of receptors that mediates the pathophysiology of olfactory dysfunction reflects the many ways in which anosmia can be therapeutically managed.

Single-cell transcriptomics identifies aberrant glomerular angiogenic signalling in the early stages of WT1 kidney disease.

WT1 encodes a podocyte transcription factor whose variants can cause an untreatable glomerular disease in early childhood. Although WT1 regulates many podocyte genes, it is poorly understood which of them are initiators in disease and how they subsequently influence other cell-types in the glomerulus. We hypothesised that this could be resolved using single-cell RNA sequencing (scRNA-seq) and ligand-receptor analysis to profile glomerular cell-cell communication during the early stages of disease in mice harbouring an orthologous human mutation in WT1 (Wt1R394W/+). Podocytes were the most dysregulated cell-type in the early stages of Wt1R394W/+ disease, with disrupted angiogenic signalling between podocytes and the endothelium, including the significant downregulation of transcripts for the vascular factors Vegfa and Nrp1. These signalling changes preceded glomerular endothelial cell loss in advancing disease, a feature also observed in biopsy samples from human WT1 glomerulopathies. Addition of conditioned medium from murine Wt1R394W/+ primary podocytes to wild-type glomerular endothelial cells resulted in impaired endothelial looping and reduced vascular complexity. Despite the loss of key angiogenic molecules in Wt1R394W/+ podocytes, the pro-vascular molecule adrenomedullin was upregulated in Wt1R394W/+ podocytes and plasma and its further administration was able to rescue the impaired looping observed when glomerular endothelium was exposed to Wt1R394W/+ podocyte medium. In comparative analyses, adrenomedullin upregulation was part of a common injury signature across multiple murine and human glomerular disease datasets, whilst other gene changes were unique to WT1 disease. Collectively, our study describes a novel role for altered angiogenic signalling in the initiation of WT1 glomerulopathy. We also identify adrenomedullin as a proangiogenic factor, which despite being upregulated in early injury, offers an insufficient protective response due to the wider milieu of dampened vascular signalling that results in endothelial cell loss in later disease. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

NRP1 knockdown inhibits the invasion and migration of rhabdoid tumor of the kidney cells.

PURPOSE: The aim of this study was to detect candidate oncogenes of rhabdoid tumor of the kidney (RTK) and evaluate their roles in RTK in vitro. METHODS: An integrated analysis of messenger RNA (mRNA) and microRNA (miRNA) sequencing was performed to determine the expression profile of exosome-derived miRNAs and mRNAs in human RTK-derived cell lines and a human embryonic renal cell line. A Gene Ontology enrichment analysis was performed to analyze the functional characteristics of differentially expressed mRNAs in RTK cells. Matrigel invasion and wound-healing assays were performed to evaluate the cell invasion and migration abilities. RESULTS: Forty mRNAs were highly expressed in RTK cells targeted by exosomal miRNAs, the expression of which was lower in RTK cells than in the controls. These mRNAs were primarily related to cell adhesion. Of these mRNAs, we selected neuropilin 1 (NRP1) as a candidate oncogene because its upregulated expression is associated with a poor prognosis of several types of tumors. RTK cells in which NRP1 had been knocked down exhibited decreased invasive and migratory abilities. CONCLUSION: Our study indicates that NRP1 acts as an oncogene by promoting the invasion and migration of RTK cells and that it could serve as a therapeutic target.

Peptidomimetic inhibitors of the VEGF-A165/NRP-1 complex obtained by modification of the C-terminal arginine.

Inhibitors of the interaction between Neuropilin-1 (NRP-1) and Vascular Endothelial Growth Factor-A165 (VEGF-A165) hold significant promise as therapeutic and diagnostic agents directed against cancers overexpressing NRP-1. In our efforts in this field, a few series of strong and fairly stable peptide-like inhibitors of the general formula Lys(Har)1-Xaa2-Xaa3-Arg4 have been previously discovered. In the current work, we focused on Lys(Har)-Dap/Dab-Pro-Arg sequence. The aim was to examine whether replacing C-terminal Arg with its homologs and mimetics would yield more stable yet still potent inhibitors. Upon considering the results of modelling and other factors, ten novel analogues with Xaa4 = homoarginine (Har), 2-amino-4-guanidino-butyric acid (Agb), 2-amino-3-guanidino-propionic acid (Agp), citrulline (Cit), 4-aminomethyl-phenylalanine [Phe(4-CH2-NH2)] were designed, synthesized and evaluated. Two of the proposed modifications resulted in inhibitors with activity slightly lower [e.g. IC50 = 14.3 µM for Lys(Har)-Dab-Pro-Har and IC50 = 19.8 µM for Lys(Har)-Dab-Pro-Phe(4-CH2-NH2)] than the parent compounds [e.g. IC50 = 4.7 µM for Lys(Har)-Dab-Pro-Arg]. What was a surprise to us, the proteolytic stability depended more on position two of the sequence than on position four. The Dab2-analogues exhibited half-life times beyond 60 h. Our results build up the knowledge on the structural requirements that effective VEGF-A165/NRP-1 inhibitors should fulfil.

NRP1 antagonism as a novel therapeutic target in nasal polyps of patients with chronic rhinosinusitis.

BACKGROUND: Neuropilin-1 (NRP1) is expressed on the surface epithelium of respiratory tract and immune cells, demonstrating its possible function in regulating the immune response in airway disease. However, its role in patient with chronic rhinosinusitis (CRS) remains unknown. This study aimed to elucidate the role of NRP1 in CRS with nasal polyps (CRSwNP). METHODS: Sinonasal biopsy specimens were immunohistochemically stained to investigate NRP1 expression. Double immunofluorescence, immunoblotting, and real-time polymerase chain reaction were performed to evaluate NRP1 in primary human nasal epithelial cells (hNECs). An NRP1 inhibitor was administered to a murine nasal polyp (NP) model. RESULTS: NRP1 was highly expressed in the epithelium in patients with CRSwNP compared to nasal tissue from controls and CRS without NP patients. NRP1 and vascular endothelial growth factor were upregulated in hNECs under hypoxia. Treatment with NRP1 inhibitor (EG00229) reduced the secretion of interleukin (IL)-1ß, IL-6, IL-8, and IL-33 cytokines, as well as inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2 in hNECs. We found that NRP1 was highly expressed in the airway epithelium in the murine NP model. The group treated with the NRP1 inhibitor had significantly fewer nasal polypoid lesions and reduced accumulations of immune cells. CONCLUSIONS: These findings reveal that NRP1 is upregulated in CRS and NP epithelium, and the inhibition of NRP1 may lead to a reduction in NP growth and immune cell infiltration. Our results suggest that NRP1 inhibition could be a novel possibility for treating nasal polyposis.

SARS-CoV-2 Rapidly Infects Peripheral Sensory and Autonomic Neurons, Contributing to Central Nervous System Neuroinvasion before Viremia.

Neurological symptoms associated with COVID-19, acute and long term, suggest SARS-CoV-2 affects both the peripheral and central nervous systems (PNS/CNS). Although studies have shown olfactory and hematogenous invasion into the CNS, coinciding with neuroinflammation, little attention has been paid to susceptibility of the PNS to infection or to its contribution to CNS invasion. Here we show that sensory and autonomic neurons in the PNS are susceptible to productive infection with SARS-CoV-2 and outline physiological and molecular mechanisms mediating neuroinvasion. Our infection of K18-hACE2 mice, wild-type mice, and golden Syrian hamsters, as well as primary peripheral sensory and autonomic neuronal cultures, show viral RNA, proteins, and infectious virus in PNS neurons, satellite glial cells, and functionally connected CNS tissues. Additionally, we demonstrate, in vitro, that neuropilin-1 facilitates SARS-CoV-2 neuronal entry. SARS-CoV-2 rapidly invades the PNS prior to viremia, establishes a productive infection in peripheral neurons, and results in sensory symptoms often reported by COVID-19 patients.

Neuropilin-1 is a novel host factor modulating the entry of hepatitis B virus.

BACKGROUND & AIMS: Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the cellular receptor for hepatitis B virus (HBV). However, hepatocytes expressing NTCP exhibit varying susceptibilities to HBV infection. This study aimed to investigate whether other host factors modulate the process of HBV infection. METHODS: Liver biopsy samples obtained from children with hepatitis B were used for single-cell sequencing and susceptibility analysis. Primary human hepatocytes, HepG2-NTCP cells, and human liver chimeric mice were used to analyze the effect of candidate host factors on HBV infection. RESULTS: Single-cell sequencing and susceptibility analysis revealed a positive correlation between neuropilin-1 (NRP1) expression and HBV infection. In the HBV-infected cell model, NRP1 overexpression before HBV inoculation significantly enhanced viral attachment and internalization, and promoted viral infection in the presence of NTCP. Mechanistic studies indicated that NRP1 formed a complex with LHBs and NTCP. The NRP1 b domain mediated its interaction with conserved arginine residues at positions 88 and 92 in the preS1 domain of the HBV envelope protein LHBs. This NRP1-preS1 interaction subsequently promoted the binding of preS1 to NTCP, facilitating viral infection. Moreover, disruption of the NRP1-preS1 interaction by the NRP1 antagonist EG00229 significantly attenuated the binding affinity between NTCP and preS1, thereby inhibiting HBV infection both in vitro and in vivo. CONCLUSIONS: Our findings indicate that NRP1 is a novel host factor for HBV infection, which interacts with preS1 and NTCP to modulate HBV entry into hepatocytes. IMPACT AND IMPLICATIONS: HBV infection is a global public health problem, but the understanding of the early infection process of HBV remains limited. Through single-cell sequencing, we identified a novel host factor, NRP1, which modulates HBV entry by interacting with HBV preS1 and NTCP. Moreover, antagonists targeting NRP1 can inhibit HBV infection both in vitro and in vivo. This study could further advance our comprehension of the early infection process of HBV.

NRP1 promotes osteo/odontogenic differentiation via shroom3 in dental pulp stem cells.

Neuropilin-1 (NRP1) is a single transmembrane glycoprotein involved in a variety of physiological events. However, the exact mechanisms by which NRP1 regulates dental pulp stem cells (DPSCs) to differentiate toward an osteo/odontogenic phenotype are poorly understood. Here, we determined the significantly increased expression of full-length NRP1 and glycosaminoglycan (GAG)-modified NRP1 during osteo/odontogenesis in DPSCs. NRP1 was confirmed to promote alkaline phosphatase (ALP) activity, mineralized nodule deposition, protein and mRNA expression of Runx2, DSPP and DMP1 in DPSCs via the loss-of-function and gain-of-function approaches. Further, a non-GAG-modified NRP1 mutant (NRP1 S612A) was generated and the suppression of osteo/odontogenic differentiation was observed in the NRP1 S612A overexpression cells. Knockdown of the adaptor protein shroom3 resulted in the inhibition of osteo/odontogenesis. The protein-protein interaction network, the protein-protein docking and confocal analyses indicated the interactions between NRP1 and shroom3. Furthermore, immunoprecipitation followed by western analysis confirmed the binding of NRP1 to shroom3, but overexpression of NRP1 S612A greatly influenced the recruitment of shroom3 by NRP1. These results provide strong evidence that NRP1 is a critical regulator for osteo/odontogenesis through interacting with shroom3. Moreover, our results indicate that NRP1 S612A attenuates osteo/odontogenesis, suggesting that GAG modification is essential for NRP1 in DPSCs.

Protease-activated CendR peptides targeting tenascin-C: mitigating off-target tissue accumulation.

To achieve precision and selectivity, anticancer compounds and nanoparticles (NPs) can be targeted with affinity ligands that engage with malignancy-associated molecules in the blood vessels. While tumor-penetrating C-end Rule (CendR) peptides hold promise for precision tumor delivery, C-terminally exposed CendR peptides can accumulate undesirably in non-malignant tissues expressing neuropilin-1 (NRP-1), such as the lungs. One example of such promiscuous peptides is PL3 (sequence: AGRGRLVR), a peptide that engages with NRP-1 through its C-terminal CendR element, RLVR.Here, we report the development of PL3 derivatives that bind to NRP-1 only after proteolytic processing by urokinase-type plasminogen activator (uPA), while maintaining binding to the other receptor of the peptide, the C-domain of tenascin-C (TNC-C). Through a rational design approach and screening of a uPA-treated peptide-phage library (PL3 peptide followed by four random amino acids) on the recombinant NRP-1, derivatives of the PL3 peptide capable of binding to NRP-1 only post-uPA processing were successfully identified. In vitro cleavage, binding, and internalization assays, along with in vivo biodistribution studies in orthotopic glioblastoma-bearing mice, confirmed the efficacy of two novel peptides, PL3uCendR (AGRGRLVR↓SAGGSVA) and SKLG (AGRGRLVR↓SKLG), which exhibit uPA-dependent binding to NRP-1, reducing off-target binding to healthy NRP-1-expressing tissues. Our study not only unveils novel uPA-dependent TNC-C targeting CendR peptides but also introduces a broader paradigm and establishes a technology for screening proteolytically activated tumor-penetrating peptides.

Identification of z-axis filopodia in growth cones using super-resolution microscopy.

A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three-dimensional-structured illumination microscopy, a type of super-resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z-axis ("z-filopodia") within the growth cone. Z-filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z-filopodia formation and dynamics were regulated by actin-regulatory proteins, such as vasodilator-stimulated phosphoprotein, fascin, and cofilin. Chromophore-assisted laser inactivation of cofilin induced the rapid turnover of z-filopodia. An axon guidance receptor, neuropilin-1, was concentrated in z-filopodia and was transported together with them, whereas its ligand, semaphorin-3A, was selectively bound to them. Membrane domains associated with z-filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin-1. The results suggest that z-filopodia have unique turnover properties, and unlike xy-filopodia, do not function as force-generating structures for axon extension.

The crosstalk between neuropilin-1 and tumor necrosis factor-α in endothelial cells.

Tumor necrosis factor-α (TNFα) is a master cytokine which induces expression of chemokines and adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in endothelial cells to initiate the vascular inflammatory response. In this study, we identified neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, as a modulator of TNFα-induced inflammatory response of endothelial cells. NRP1 shRNA expression suppressed TNFα-stimulated leukocyte adhesion and expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs). Likewise, it reduced TNFα-induced phosphorylation of MAPK p38 but did not significantly affect other TNF-induced signaling pathways, such as the classical NFκB and the AKT pathway. Immunofluorescent staining demonstrated co-localization of NRP1 with the two receptors of TNF, TNFR1 and TNFR2. Co-immunoprecipitation further confirmed that NRP1 was in the same protein complex or membrane compartment as TNFR1 and TNFR2, respectively. Modulation of NRP1 expression, however, neither affected TNFR levels in the cell membrane nor the receptor binding affinities of TNFα. Although a direct interface between NRP1 and TNFα/TNFR1 appeared possible from a protein docking model, a direct interaction was not supported by binding assays in cell-free microplates and cultured cells. Furthermore, TNFα was shown to downregulate NRP1 in a time-dependent manner through TNFR1-NFκB pathway in HUVECs. Taken together, our study reveals a novel reciprocal crosstalk between NRP1 and TNFα in vascular endothelial cells.

Targeting the Neuropilin-1 receptor with Ovatodiolide and progress in using periodontal ligament organoids for COVID-19 research and therapy.

The discovery of SARS-CoV-2 RNA in the periodontal tissues of patients who tested positive for COVID-19, 24 days post the initial symptom onset, indicates the oral cavity could serve as a viral reservoir. This research aims to investigate the antiviral capabilities of Ovatodiolide, introducing a novel periodontal ligament organoid model for the study of SARS-CoV-2. We have successfully established a reliable and expandable organoid culture from the human periodontal ligament, showcasing characteristics typical of epithelial stem cells. This organoid model enables us to delve into the lesser-known aspects of dental epithelial stem cell biology and their interactions with viruses and oral tissues. We conducted a series of in vitro and ex vivo studies to examine the inhibitory impacts of Ova on SARS-CoV-2. Our findings indicate that Ovatodiolide molecules can bind effectively to the NRP1 active domain. Our study identifies potential interaction sites for Ovatodiolide (OVA) within the b1 domain of the NRP1 receptor. We generated point mutations at this site, resulting in three variants: Y25A, T44A, and a double mutation Y25A/T44A. While these mutations did not alter the binding activity of the spike protein, they did impact the concentration of OVA required for inhibition. The inhibitory concentrations for these variants are 15 µM for Y25A, 15.2 µM for T44A, and 25 µM for the double mutant Y25A/T44A. In addition, in vitro inhibition experiments demonstrate that the EC50 of Ova against the main protease (Mpro) of the SARS-CoV-2 virus is 7.316 µM. Our in vitro studies and the use of the periodontal ligament organoid model highlight Ovatodiolide's potential as a small molecule therapeutic agent that impedes the virus's ability to bind to the Neuropilin-1 receptor on host cells. The research uncovers various pathways and biochemical strategies through which Ovatodiolide may function as an effective antiviral small molecule drug.

Neuropilin-1 sex-dependently modulates inflammatory, angiogenic and osteogenic phenotypes in the calcifying valve interstitial cell.

The pathological mechanisms underlying the sex-dependent presentation of calcific aortic stenosis (AS) remain poorly understood. We aim to analyse sex-specific responses of valve interstitial cells (VICs) to calcific environments and to identify new pathological and potentially druggable targets. First, VICs from stenotic patients were modelled using pro-calcifying media (HP). Both male and female VICs were inflamed upon calcific HP challenge, although the inflammatory response was higher in female VICs. The osteogenic and calcification responses were higher in male VICs. To identify new players involved in the responses to HP, proteomics analyses were performed on additional calcifying VICs. Neuropilin-1 (NRP-1) was significantly up-regulated in male calcifying VICs and that was confirmed in aortic valves (AVs), especially nearby neovessels and calcifications. Regardless of the sex, NRP-1 expression was correlated to inflammation, angiogenesis and osteogenic markers, but with stronger associations in male AVs. To further evidence the role of NRP-1, in vitro experiments of silencing or supplementation with soluble NRP-1 (sNRP-1) were performed. NRP-1 silencing or addition of sNRP-1 reduced/mended the expression of any sex-specific response triggered by HP. Moreover, NRP-1 regulation contributed to significantly diminish the baseline enhanced expression of pro-inflammatory, pro-angiogenic and pro-osteogenic markers mainly in male VICs. Validation studies were conducted in stenotic AVs. In summary, pharmacologic targeting of NRP-1 could be used to target sex-specific phenotypes in AS as well as to exert protective effects by reducing the basal expression of pathogenic markers only in male VICs.

Construction of a novel cancer-associated fibroblast-related signature to predict clinical outcome and immune response in cervical cancer.

This study developed a prognostic signature for cervical cancer using transcriptome profiling and clinical data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and TISCH database, focusing on cancer-associated fibroblasts (CAFs). Through LASSO Cox regression and integrated bioinformatics analyses, we identified 144 differentially expressed genes (DEGs) related to CAFs, from which an 11-gene CAF-related signature (CAFRSig) was constructed. The CAFRSig effectively stratified patients into high- and low-risk categories, demonstrating significant prognostic capability in predicting overall survival. Gene ontology (GO) and gene set variation analysis (GSVA) linked the DEGs to crucial pathways in tumor malignancy, immune response, and fatty acid metabolism. The immune landscape analysis, utilizing the TIMER platform and CIBERSORT algorithm, revealed a positive correlation between immune cell effector functions and CAFRSig scores, highlighting the model's potential to identify patients likely to respond to immune checkpoint blockade (ICB) therapies. Furthermore, neuropilin 1 (NRP1), a key gene in the CAFRSig, was upregulated in cervical cancer tissues and associated with disease progression and differentiation. The downregulation of NRP1 curbed cell proliferation and influenced the epithelial-mesenchymal transition (EMT), implicating the PI3K/AKT pathway and modulating PD-L1 expression. This comprehensive analysis establishes a robust prognostic signature based on CAF-related genes, offering valuable insights for optimizing therapeutic strategies in cervical cancer management.

Differential modulation of cell morphology, migration, and Neuropilin-1 expression in cancer and non-cancer cell lines by substrate stiffness.

Physical changes in the tumor microenvironment, such as increased stiffness, regulate cancer hallmarks and play an essential role in gene expression, cell morphology, migration, and malignancy. However, the response of cancer cells to stiffness is not homogeneous and varies depending on the cell type and its mechanosensitivity. In this study, we investigated the differential responses of cervical (HeLa) and prostate (PC-3) cancer cell lines, as well as non-tumoral cell lines (HEK293 and HPrEC), to stiffness using polyacrylamide hydrogels mimicking normal and tumoral tissues. We analyzed cell morphology, migration, and the expression of neuropilin 1 (NRP1), a receptor involved in angiogenesis, cell migration, and extracellular matrix remodeling, known to be associated with cancer progression and poor prognosis. Our findings reveal that NRP1 expression increases on substrates mimicking the high stiffness characteristic of tumoral tissue in the non-tumoral cell lines HPrEC and HEK293. Conversely, in tumoral PC-3 cells, stiffness resembling normal prostate tissue induces an earlier and more sustained expression of NRP1. Furthermore, we observed that stiffness influences cell spreading, pseudopodia formation, and the mode of cell protrusion during migration. Soft substrates predominantly trigger bleb cell protrusion, while pseudopodia protrusions increase on substrates mimicking normal and tumor-like stiffnesses in HPrEC cells compared to PC-3 cells. Stiffer substrates also enhance the percentage of migratory cells, as well as their velocity and total displacement, in both non-tumoral and tumoral prostate cells. However, they only improve the persistence of migration in tumoral PC-3 cells. Moreover, we found that NRP1 co-localizes with actin, and its suppression impairs tumoral PC-3 spreading while decreasing pseudopodia protrusion mode. Our results suggest that the modulation of NRP1 expression by the stiffness can be a feedback loop to promote malignancy in non-tumoral and cancer cells, contingent upon the mechanosensitivity of the cells.

Identification of an osteopontin-derived peptide that binds neuropilin-1 and activates vascular repair responses and angiogenesis.

The osteopontin-derived peptide FOL-005 stimulates hair growth. Using ligand-receptor glyco-capture technology we identified neuropilin-1 (NRP-1), a known co-receptor for vascular endothelial growth factor (VEGF) receptors, as the most probable receptor for FOL-005 and the more stable analogue FOL-026. X-ray diffraction and microscale thermophoresis analysis revealed that FOL-026 shares binding site with VEGF in the NRP-1 b1-subdomain. Stimulation of human umbilical vein endothelial cells with FOL-026 resulted in phosphorylation of VEGFR-2, ERK1/2 and AKT, increased cell growth and migration, stimulation of endothelial tube formation and inhibition of apoptosis in vitro. FOL-026 also promoted angiogenesis in vivo as assessed by subcutaneous Matrigel plug and hind limb ischemia models. NRP-1 knock-down or treatment of NRP-1 antagonist EG00229 blocked the stimulatory effects of FOL-026 on endothelial cells. Exposure of human coronary artery smooth muscle cells to FOL-026 stimulated cell growth, migration, inhibited apoptosis, and induced VEGF gene expression and VEGFR-2/AKT phosphorylation by an NRP-1-dependent mechanism. RNA sequencing showed that FOL-026 activated pathways involved in tissue repair. These findings identify NRP-1 as the receptor for FOL-026 and show that its biological effects mimic that of growth factors binding to the VEGF receptor family. They also suggest that FOL-026 may have therapeutical potential in conditions that require vascular repair and/or enhanced angiogenesis.

Soluble Neuropilin-1 Is Elevated in Sepsis and Correlates with Organ Dysfunction and Long-Term Mortality in Critical Illness.

Critical illness and sepsis may cause organ failure and are recognized as mortality drivers in hospitalized patients. Neuropilin-1 (NRP-1) is a multifaceted transmembrane protein involved in the primary immune response and is expressed in immune cells such as T and dendritic cells. The soluble form of NRP-1 (sNRP-1) acts as an antagonist to NRP-1 by scavenging its ligands. The aim of this study was to determine the value of sNRP-1 as a biomarker in critical illness and sepsis. We enrolled 180 critically ill patients admitted to a medical intensive care unit and measured serum sNRP-1 concentrations at admission, comparing them to 48 healthy individuals. Critically ill and septic patients showed higher levels of sNRP-1 compared to healthy controls (median of 2.47 vs. 1.70 nmol/L, p < 0.001). Moreover, sNRP-1 was also elevated in patients with sepsis compared to other critical illness (2.60 vs. 2.13 nmol/L, p = 0.01), irrespective of disease severity or organ failure. In critically ill patients, sNRP-1 is positively correlated with markers of kidney and hepatic dysfunction. Most notably, critically ill patients not surviving in the long term (one year after admission) showed higher concentrations of sNRP-1 at the time of ICU admission (p = 0.036), with this association being dependent on the presence of organ failure. Critically ill and septic patients exhibit higher serum concentrations of circulating sNRP-1, which correlates to organ failure, particularly hepatic and kidney dysfunction.

NRP1 AS A POTENTIAL MOLECULAR TARGET FOR MEDULOBLASTOMA / NRP1 COMO POTENCIAL ALVO MOLECULAR PARA MEDULOBLASTOMA

Introdução : O meduloblastoma é um tumor maligno, altamente agressivo e de rápido crescimento que surge no cerebelo ou no assoalho do quarto ventrículo e tronco encefálico, principalmente em crianças. Mesmo com os avanços na terapia, a morbidade e a mortalidade continuam sendo um grande desafio. Portanto, novos tratamentos são necessários para reduzir esses resultados. Objetivo : Revisar a relação NRP1 (neuropilina 1) e meduloblastoma como potencial alvo terapêutico e, também, com sobrevida global. Method: This is a narrative review carried out in the PubMed and Scielo databases. The search used the following keywords: "neuropilins, medulloblastoma, brain tumors, pediatrics". The inclusion criteria were review articles, experimental studies, pre-clinical and clinical research, in English and Portuguese, and available in full text. The selected articles were analyzed based on the technologies covered, future perspectives and challenges mentioned, diseases mentioned and the central idea of the article. Result: 30 articles were included. Conclusion: Medulloblastomas have high transcriptional levels of neuropilin (NRP1) and their low levels are related to lower overall survival, especially in SHH. In this sense, NRP1 and its complex action system appear as a potential target for oncological therapies for brain tumors.

Introdução : Meduloblastoma é um tumor maligno, altamente agressivo e de rápido crescimento que surge no cerebelo ou no assoalho do quarto ventrículo e tronco cerebral, especialmente em crianças. Mesmo com os avanços na terapia, a morbimortalidade permanece um grande desafio. Por isso, novos tratamentos são necessários para reduzir esses estágios. Objetivo : Revisar a relação NRP1 (neuropilina 1) e meduloblastoma como potencial alvo terapêutico e, também, com sobrevida global. Método : Trata-se de revisão narrativa realizada nas bases de dados PubMed e Scielo. A busca utilizou as seguintes palavras-chave: " neuropilinas, meduloblastoma, tumores malignos, pediatria ". Os critérios de inclusão foram artigos de revisão, estudos experimentais, pesquisas pré-clínicas e clínicas, em inglês e português, e disponíveis em texto completo. Os artigos selecionados foram analisados ​​com base nas tecnologias abordadas, perspectivas futuras e desafios conceituais, doenças referidas e ideia central do artigo. Resultado : Foram incluídos 30 artigos Conclusão : Os meduloblastomas possuem altos níveis transcricionais de neuropilina (NRP1) e seus baixos níveis relacionados com a menor sobrevida global, especialmente nos SHH. Nesse sentido, o NRP1 e seu complexo sistema de atuação aparecem como alvo potencial de terapias oncológicas para tumores específicos.

Pharmacological inhibition of PDGF-C/neuropilin-1 interaction: A novel strategy to reduce melanoma metastatic potential.

Activation of neuropilin-1 (NRP-1) by platelet derived growth factor (PDGF)-C sustains melanoma invasiveness. Therefore, in the search of novel agents capable of reducing melanoma spreading, PDGF-C/NRP-1 interaction was investigated as a potential druggable target. Since the PDGF-C region involved in NRP-1 binding is not yet known, based on the sequence and structural homology between PDGF-C and vascular endothelial growth factor-A (VEGF-A), we hypothesized that the NRP-1 b1 domain region involved in the interaction with VEGF-A might also be required for PDGF-C binding. Hence, this region was selected from the protein crystal structure and used as target in the molecular docking procedure. In the following virtual screening, compounds from a DrugBank database were used as query ligands to identify agents potentially capable of disrupting NRP-1/PDGF-C interaction. Among the top 45 candidates with the highest affinity, five drugs were selected based on the safety profile, lack of hormonal effects, and current availability in the market: the antipsychotic pimozide, antidiabetic gliclazide, antiallergic cromolyn sodium, anticancer tyrosine kinase inhibitor entrectinib, and antihistamine azelastine. Analysis of drug influence on PDGF-C in vitro binding to NRP-1 and PDGF-C induced migration of human melanoma cells expressing NRP-1, indicated gliclazide and entrectinib as the most specific agents that were active at clinically achievable and non-toxic concentrations. Both drugs also reverted PDGF-C ability to stimulate extracellular matrix invasion by melanoma cells resistant to BRAF inhibitors. The inhibitory effect on tumor cell motility involved a decrease of p130Cas phosphorylation, a signal transduction pathway activated by PDGF-C-mediated stimulation of NRP-1.

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.