Interferon-γ Drives Treg Fragility to Promote Anti-tumor Immunity.

Regulatory T cells (Tregs) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral Treg stability and function but is dispensable for peripheral immune tolerance. Treg-restricted Nrp1 deletion results in profound tumor resistance due to Treg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of Treg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1+ Tregs correlates with poor prognosis in melanoma and head and neck squamous cell carcinoma. Using a mouse model of melanoma where Nrp1-deficient (Nrp1-/-) and wild-type (Nrp1+/+) Tregs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1-/- Tregs produce interferon-γ (IFNγ), which drives the fragility of surrounding wild-type Tregs, boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFNγ-induced Treg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting Treg fragility may be efficacious.

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection.

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model, we further demonstrate that pDCs, while not supporting SARS-CoV-2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL-6, IL-8, CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways, we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL-6 response, suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection.

High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

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.

ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

Dual effects of targeting neuropilin-1 in lenvatinib-resistant hepatocellular carcinoma: inhibition of tumor growth and angiogenesis.

In the era of immunotherapy, lenvatinib (LEN) still holds an important position in the sequential treatment of advanced hepatocellular carcinoma (HCC). However, the sustained therapeutic effect of LEN is not sufficient, and there is a need to address the development of resistance. Neuropilin-1 (NRP1) is known to act as a coreceptor for epidermal growth factor receptor (EGFR), Met, and vascular endothelial growth factor receptor 2 (VEGFR2), which have been reported to be involved in LEN resistance. In this study, we used cell culture and in vivo xenograft models to evaluate the contribution of NRP1 in the acquisition of LEN resistance in HCC as well as the potential of NRP1 as a therapeutic target. LEN resistance increased EGF/EGFR and hepatocyte growth factor (HGF)/Met signaling in liver cancer cells and VEGFA/VEGFR2 and HGF/Met signaling in vascular endothelial cells, thereby promoting cell proliferation, cell migration, and angiogenesis. We found that activation of NRP1 is essential for the enhancement of these signaling. In addition, NRP1 inhibition combined with LEN therapy synergistically improved the antitumor effects against LEN-resistant HCC, indicating that NRP1 is an attractive therapeutic target.NEW & NOTEWORTHY We demonstrated that neuropilin-1 (NRP1) was an essential coreceptor mediating the activation of multiple signaling pathways in the acquisition of resistance to lenvatinib (LEN) in HCC. The addition of NRP1 inhibition to LEN had a synergistic antitumor effect on LEN-resistant HCC in culture and in vivo xenograft models.

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.

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.

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 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 (large hepatitis B surface proteins) and NTCP. The NRP1 b domain mediated its interaction with conserved arginine residues at positions 88 and 92 in the preS1 domain of 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 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.

Preclinical evaluation of 68 Ga-labeled peptide CK2 for PET imaging of NRP-1 expression in vivo.

PURPOSE: Neuropilin-1 (NRP-1) is a multifunctional protein involved in a variety of biological processes such as angiogenesis, tumorigenesis and immunomodulation. It was usually overexpressed in many cancer cell lines and correlated with poor prognosis of breast cancer. Positron emission tomography (PET) is an advanced imaging technique for detecting the function and metabolism of tumor-associated molecules in real time, dynamically, quantitatively and noninvasively. To improve the level of early diagnosis and evaluate the prognosis of breast cancer, an NRP-1 targeting peptide-based tracer [68 Ga]Ga-NOTA-PEG4-CK2 was designed to sensitively and specifically detect the NRP-1 expression in vivo via PET imaging. METHODS: In silico modeling and microscale thermophoresis (MST) assay were carried out to design the NRP-1 targeting peptide NOTA-PEG4-CK2, and it was further radiolabeled with 68 Ga to prepare the tracer [68 Ga]Ga-NOTA-PEG4-CK2. The radiochemical yield (RCY), radiochemical purity (RCP), molar activity (Am), lipid-water partition coefficient (Log P) and stability of [68 Ga]Ga-NOTA-PEG4-CK2 were assessed. The targeting specificity of the tracer for NRP-1 was investigated by in vitro cellular uptake assay and in vivo PET imaging as well as blocking studies. The sensitivity of the tracer in monitoring the dynamic changes of NRP-1 expression induced by chemical drug was also investigated in vitro and in vivo. Ex vivo biodistribution, autoradiography, western blot, and immunofluorescence staining were also performed to study the specificity of [68 Ga]Ga-NOTA-PEG4-CK2 for NRP-1. RESULTS: [68 Ga]Ga-NOTA-PEG4-CK2 was designed and synthesized with high RCY (> 98%), high stability (RCP > 95%) and high affinity to NRP-1 (KD = 25.39 ± 1.65 nM). In vitro cellular uptake assay showed that the tracer [68 Ga]Ga-NOTA-PEG4-CK2 can specifically bind to NRP-1 positive cancer cells MDA-MB-231 (1.04 ± 0.04% at 2 h) rather than NRP-1 negative cancer cells NCI-H1299 (0.43 ± 0.05%). In vivo PET imaging showed the maximum tumor uptake of [68 Ga]Ga-NOTA-PEG4-CK2 in MDA-MB-231 xenografts (4.16 ± 0.67%ID/mL) was significantly higher than that in NCI-H1299 xenografts (1.03 ± 0.19%ID/mL) at 10 min post injection, and the former exhibited higher tumor-to-muscle uptake ratio (5.22 ± 0.18) than the latter (1.07 ± 0.27) at 60 min post injection. MDA-MB-231 xenografts pretreated with nonradioactive precursor NOTA-PEG4-CK2 showed little tumor uptake of [68 Ga]Ga-NOTA-PEG4-CK2 (1.67 ± 0.38%ID/mL at 10 min post injection). Both cellular uptake assay and PET imaging revealed that NRP-1 expression in breast cancer MDA-MB-231 could be effectively suppressed by SB-203580 treatment and can be sensitively detected by [68 Ga]Ga-NOTA-PEG4-CK2. Ex vivo analysis also proved the high specificity and sensitivity of [68 Ga]Ga-NOTA-PEG4-CK2 for NRP-1 expression in MDA-MB-231 xenografts. CONCLUSION: A promising NRP-1 targeting PET tracer [68 Ga]Ga-NOTA-PEG4-CK2 was successfully prepared. It showed remarkable specificity and sensitivity in monitoring the dynamic changes of NRP-1 expression. Hence, it could provide valuable information for early diagnosis of NRP-1 relevant cancers and evaluating the prognosis of cancer patients.

O-glycan structures in apo(a) subunit of human lipoprotein(a) suppresses the pro-angiogenic activity of galectin-1 on human umbilical vein endothelial cells.

Apolipoprotein(a) [apo(a)] is a highly polymorphic O-glycoprotein circulating in human plasma as lipoprotein(a) [Lp(a)]. The O-glycan structures of apo(a) subunit of Lp(a) serve as strong ligands of galectin-1, an O-glycan binding pro-angiogenic lectin abundantly expressed in placental vascular tissues. But the pathophysiological significance of apo(a)-galectin-1 binding is not yet been revealed. Carbohydrate-dependent binding of galectin-1 to another O-glycoprotein, neuropilin-1 (NRP-1) on endothelial cells activates vascular endothelial growth factor receptor 2 (VEGFR2) and mitogen-activated protein kinase (MAPK) signaling. Using apo(a), isolated from human plasma, we demonstrated the potential of the O-glycan structures of apo(a) in Lp(a) to inhibit angiogenic properties such as proliferation, migration, and tube-formation in human umbilical vein endothelial cells (HUVECs) as well as neovascularization in chick chorioallantoic membrane. Further, in vitro protein-protein interaction studies have confirmed apo(a) as a superior ligand to NRP-1 for galectin-1 binding. We also demonstrated that the protein levels of galectin-1, NRP-1, VEGFR2, and downstream proteins in MAPK signaling were reduced in HUVECs in the presence of apo(a) with intact O-glycan structures compared to that of de-O-glycosylated apo(a). In conclusion, our study shows that apo(a)-linked O-glycans prevent the binding of galectin-1 to NRP-1 leading to the inhibition of galectin-1/neuropilin-1/VEGFR2/MAPK-mediated angiogenic signaling pathway in endothelial cells. As higher plasma Lp(a) level in women is an independent risk factor for pre-eclamsia, a pregnancy-associated vascular complication, we propose that apo(a) O-glycans-mediated inhibition of the pro-angiogenic activity of galectin-1 may be one of the underlying molecular mechanism of pathogenesis of Lp(a) in pre-eclampsia.

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.

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.

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.

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.

Expression of placental growth factor, neuropilin-1, and neuropilin-2 in primary pterygium tissue.

PURPOSE: The aim of this study was to evaluate the expression of placental growth factor (PLGF), neuropilin-1 (NP-1), and neuropilin-2 (NP-2) molecules in primary pterygium tissue compared with normal conjunctival tissue. METHODS: The records of 42 patients who underwent excision surgery with autografts for primary pterygium (pterygium group) and 20 patients who underwent conjunctival nevus excision surgery (control group) in the same period were reviewed retrospectively. The samples obtained from the pterygium tissues in the pterygium group and the clean conjunctival tissues adjacent to the nevus in the control group were collected from the archive. Immunohistochemical stains of the primary antibodies-1/100 diluted PLGF, NP-1, and NP-2 (Abcam Cambridge Science Park, UK)-were applied to all groups. Staining intensities and the percentage of positive cells in epithelial, endothelial, stromal, and inflammatory cells were analyzed by an experienced pathologist. RESULTS: The positivity rates of PLGF and NP-2 expression in epithelial, endothelial, stromal, and inflammatory cells were found to be higher in the pterygium group than in the control group (PLGF: p < 0.001, p < 0.001, p = 0.001, and p < 0.001, respectively; NP-2: p < 0.001 for all). Staining intensities for PLGF and NP-2 were higher in the pterygium group than in the control group (PLGF: p < 0.001, p < 0.001, p = 0.005, and p < 0.001, respectively; NP-2: p < 0.001, p < 0.001, p = 0.001, and p < 0.001, respectively). However, no significant differences were found in any cell type in terms of NP-1 expression positivity rates (p = 0.730, p = 0.121, p = 0.524, and p = 0.624, respectively) or staining intensity (p = 0.716, p = 0.147, p = 0.147, and p = 0.780, respectively). CONCLUSION: PLGF and NP-2 levels were found to be higher in pterygium tissue, while there was no difference in NP-1. These results indicate the possible roles of NP-2 and PLGF in primary pterygium.

Neuropilin-1 enhances temozolomide resistance in glioblastoma via the STAT1/p53/p21 axis.

Glioblastoma (GBM) is the most prevalent primary intracranial tumor. Temozolomide (TMZ) is the first-line chemotherapy for GBM. Nonetheless, the development of TMZ resistance has become a main cause of treatment failure in GBM patients. Evidence suggests that neuropilin-1 (NRP-1) silencing can attenuate GBM cell resistance to TMZ. This study aims to determine potential mechanisms by which NRP-1 affects TMZ resistance in GBM. The parental U251 and LN229 GBM cells were exposed to increasing concentrations of TMZ to construct TMZ-resistant GBM cells (U251/TMZ, LN229/TMZ). BALB/c nude mice were injected with U251/TMZ cells to establish the xenograft mouse model. Functional experiments were carried out to examine NRP-1 functions. Western blotting and real-time quantitative polymerase chain reaction were used to evaluate molecular protein and mRNA expression, respectively. Immunohistochemical staining showed NRP-1 and STAT1 expression in mouse tumors. The results showed that NRP-1 was highly expressed in TMZ-resistant cells. Moreover, knocking down NRP-1 attenuated the TMZ resistance of U251/TMZ cells, while upregulating NRP-1 enhanced TMZ resistance of the parental cells. NRP-1 silencing elevated GBM cell sensitivity to TMZ in tumor-bearing mice. Depleting NRP-1 reduced STAT1, p53, and p21 expression in U251/TMZ cells. STAT1 depletion offset NRP-1 silencing evoked attenuation of GBM cell resistance to TMZ. Collectively, our study reveals that NRP-1 enhances TMZ resistance in GBM possibly by regulating the STAT1/p53/p21 axis.

CMT2N-causing aminoacylation domain mutants enable Nrp1 interaction with AlaRS.

Through dominant mutations, aminoacyl-tRNA synthetases constitute the largest protein family linked to Charcot-Marie-Tooth disease (CMT). An example is CMT subtype 2N (CMT2N), caused by individual mutations spread out in AlaRS, including three in the aminoacylation domain, thereby suggesting a role for a tRNA-charging defect. However, here we found that two are aminoacylation defective but that the most widely distributed R329H is normal as a purified protein in vitro and in unfractionated patient cell samples. Remarkably, in contrast to wild-type (WT) AlaRS, all three mutant proteins gained the ability to interact with neuropilin 1 (Nrp1), the receptor previously linked to CMT pathogenesis in GlyRS. The aberrant AlaRS-Nrp1 interaction is further confirmed in patient samples carrying the R329H mutation. However, CMT2N mutations outside the aminoacylation domain do not induce the Nrp1 interaction. Detailed biochemical and biophysical investigations, including X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange (HDX), switchSENSE hydrodynamic diameter determinations, and protease digestions reveal a mutation-induced structural loosening of the aminoacylation domain that correlates with the Nrp1 interaction. The b1b2 domains of Nrp1 are responsible for the interaction with R329H AlaRS. The results suggest Nrp1 is more broadly associated with CMT-associated members of the tRNA synthetase family. Moreover, we revealed a distinct structural loosening effect induced by a mutation in the editing domain and a lack of conformational impact with C-Ala domain mutations, indicating mutations in the same protein may cause neuropathy through different mechanisms. Our results show that, as with other CMT-associated tRNA synthetases, aminoacylation per se is not relevant to the pathology.