Neuropilin-1 Research Articles

The role of neuropilin-1 (NRP1) in the development of SARS-CoV-2 infection

Представлен обзор литературы о роли нейропилина-1 в развитии инфекции SARS-CoV-2 и поиск вероятных связей полиморфных вариантов гена NRP1 c SARS-CoV-2. В данном обзоре приведены характеристики полиморфных вариантов гена NRP1, которые демонстрируют возможность их связи с течением инфекции SARS-CoV-2.

A Novel circUBR4/miR-491-5p/NRP2 ceRNA Network Regulates Oxidized Low-density Lipoprotein-induced Proliferation and Migration in Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) play critical roles in the progression of atherosclerosis. Circular RNA (circRNA) ubiquitin protein ligase E3 component n-recognin 4 (circUBR4) has been shown to regulate VSMC migration and proliferation. In this study, we sought to identify the mechanism in the regulation of circUBR4. CircUBR4, microRNA (miR)-491-5p, and Neuropilin-2 (NRP2) were quantified by quantitative real-time polymerase chain reaction (PCR) and western blot. Cell proliferation was evaluated by Cell Counting Kit-8 and 5-Ethynyl-2'-Deoxyuridine assays. Cell migration was examined by wound-healing and transwell invasion assays. The direct relationship between miR-491-5p and circUBR4 or NRP2 was validated by dual-luciferase reporter and RNA immunoprecipitation assays. Our data indicated that in VSMCs, ox-LDL induced circUBR4 expression. Silencing endogenous circUBR4 attenuated VSMC proliferation and migration induced by ox-LDL. Mechanistically, circUBR4 targeted miR-491-5p by pairing to miR-491-5p. Moreover, miR-491-5p was identified as a downstream mediator of circUBR4 function in ox-LDL-treated VSMCs. NRP2 was a direct target of miR-491-5p, and circUBR4 acted as a competing endogenous RNA for miR-491-5p to regulate NRP2 expression. In addition, NRP2 was a functionally downstream effector of miR-491-5p in regulating ox-LDL-evoked VSMC proliferation and migration. Our findings identify a new competing endogenous RNA network, the circUBR4/miR-491-5p/NRP2 axis, for the regulation of circUBR4 in VSMC migration and proliferation.

Immunofluorescence Studies on the Expression of the SARS-CoV-2 Receptors in Human Term Placenta

Until September 2021, the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2; COVID-19) pandemic caused over 217 million infections and over 4.5 million deaths. In pregnant women, the risk factors for the need of intensive care treatment are generally the same as in the overall population. Of note, COVID-19-positive women deliver earlier than COVID-19-negative women, and the risk for severe neonatal and perinatal morbidity and mortality is significantly higher. The probability and pathways of vertical transmission of the virus from the pregnant woman to the fetus are highly controversial. Recent data have shown that 54 (13%) of 416 neonates born to COVID-19-positive women were infected. Here, we investigated term placentas collected before the SARS-CoV-2 pandemic and studied the main COVID-19 receptors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine subtype 2 (TMPRSS2), as well as neuropilin 1 (NRP1). We performed real-time PCR and immunofluorescence on cryosections in combination with markers for syncytiotrophoblast, endothelial cells, macrophages and stromal cells. The PCR studies showed expression of both the truncated delta form of ACE2, which does not bind the COVID-19 spike protein, and the long form. The ACE2 antibody used does not distinguish between the two forms. We did not observe expression of the canonical SARS-CoV-2 entry machinery on syncytio- and cytotrophoblast. ACE2 and TMPRSS2 are co-expressed in a subpopulation of stromal cells, which in part are CD68-positive macrophages. NRP1 is localized to endothelial cells. In sum, the term placenta is not an organ that directly favors vertical transmission of COVID-19; however, microtraumas and placentitis may weaken its barrier function.

Effects of Weight Loss on Adipose and Muscular Neuropilin 1 mRNA Expression in Obesity: Potential Implication in SARS-CoV-2 Infections?

Introduction: Neuropilin 1 (NRP-1) is a novel co-receptor promoting SARS-CoV-2 infectivity. Animal data indicate a role in trans-endothelial lipid transport and storage. As human data are sparse, we aimed to assess the role of NRP-1 in 2 metabolic active tissues in human obesity and in the context of weight loss-induced short- and long-term metabolic changes. Methods: After a standardized 12-week weight reduction program, 143 subjects (age >18; body mass index ≥27 kg/m², 78% female) were randomized to a 12-month lifestyle intervention or a control group using a stratified randomization scheme. This was followed by 6-month follow-up without any intervention. Phenotyping was performed before and after weight loss, after 12-month intervention and after subsequent 6 months of follow-up. Tissue-specific insulin sensitivity was estimated by HOMA-IR (whole body and mostly driven by liver), insulin sensitivity index (ISI)_Clamp (predominantly skeletal muscle), and free fatty acid (FFA) suppression during hyperinsulinemic-euglycemic clamp (FFA_Supp) (predominantly adipose tissue). NRP-1 mRNA expression was measured in subcutaneous adipose tissue (NRP-1_AT) and skeletal muscle (NRP-1_SM) before and after weight loss. Results: NRP-1 was highly expressed in adipose tissue (7,893 [7,303–8,536] counts), but neither NRP-1_AT nor NRP-1_SM were related to estimates of obesity. Higher NRP-1_AT was associated with stronger FFA_Supp (r = −0.343, p = 0.003) and a tendency to higher ISI_Clamp (r = 0.202, p = 0.085). Weight loss induced a decline of NRP-1_AT but not NRP-1_SM. This was more pronounced in subjects with stronger reduction of adipose ACE-2 mRNA expression (r = 0.250; p = 0.032) but was not associated with short- and long-term improvement of FFA_Supp and ISI_Clamp. Conclusion: NRP-1_AT is related to adipose insulin sensitivity in obesity. Weight loss-induced decline of NRP-1_AT seems not to be involved in metabolic short- and long-term improvements after weight loss. However, weight loss-induced reduction of both NRP-1_AT and ACE-2_AT indicates a lower susceptibility of adipose tissue for SARS-CoV-2 after body weight reduction.

Prevalence of intratumoral regulatory T cells expressing neuropilin-1 is associated with poorer outcomes in patients with cancer.

Despite the success of immune checkpoint blockade therapy, few strategies sufficiently overcome immunosuppression within the tumor microenvironment (TME). Targeting regulatory T cells (Tregs) is challenging, because perturbing intratumoral Treg function must be specific enough to avoid systemic inflammatory side effects. Thus, no Treg-targeted agents have proven both safe and efficacious in patients with cancer. Neuropilin-1 (NRP1) is recognized for its role in supporting intratumoral Treg function while being dispensable for peripheral homeostasis. Nonetheless, little is known about the biology of human NRP1+ Tregs and the signals that regulate NRP1 expression. Here, we report that NRP1 is preferentially expressed on intratumoral Tregs across six distinct cancer types compared to healthy donor peripheral blood [peripheral blood lymphocyte (PBL)] and site-matched, noncancer tissue. Furthermore, NRP1+ Treg prevalence is associated with reduced progression-free survival in head and neck cancer. Human NRP1+ Tregs have broad activation programs and elevated suppressive function. Unlike mouse Tregs, we demonstrate that NRP1 identifies a transient activation state of human Tregs driven by continuous T cell receptor (TCR) signaling through the mitogen-activated protein kinase pathway and interleukin-2 exposure. The prevalence of NRP1+ Tregs in patient PBL correlates with the intratumoral abundance of NRP1+ Tregs and may indicate higher disease burden. These findings support further clinical evaluation of NRP1 as a suitable therapeutic target to enhance antitumor immunity by inhibiting Treg function in the TME.

Neuropilin-2 regulates airway inflammation in a neutrophilic asthma model.

BackgroundAsthma is a heterogenous disease that can be classified into eosinophilic (type 2‐high) and noneosinophilic (type 2‐low) endotypes. The type 2‐low endotype of asthma can be characterized by the presence of neutrophilic airway inflammation that is poorly responsive to corticosteroids. Dysregulated innate immune responses to microbial products including Toll‐like receptor (TLR) ligands have been associated with the pathogenesis of neutrophilic asthma. The key molecules that regulate inflammatory responses in individuals with neutrophilic asthma remain unclear. We previously reported that the immunoregulatory receptor neuropilin‐2 (NRP2) is expressed by murine and human alveolar macrophage (AM) and suppresses lipopolysaccharide (LPS)‐induced neutrophilic airway inflammation.MethodsHere, we investigated the immunoregulatory role of NRP2 in a mouse model of neutrophilic asthma.ResultsWe found that TLR ligands, but not T helper 2 (Th2)‐promoting adjuvants, induced NRP2 expression by AM. Using an LPS‐mediated model of neutrophilic asthma, we demonstrate that NRP2 was increased in AM and other lung antigen‐presenting cells following airway challenge with antigen. Conditional deletion of NRP2 in myeloid cells exacerbated airway inflammation in a neutrophilic asthma model. In contrast, myeloid‐specific ablation of NRP2 did not affect airway inflammation in a Th2‐mediated eosinophilic asthma model. Myeloid‐specific ablation of NRP2 did not affect Th1/Th17 responses to inhaled antigens or expression of neutrophil chemokines but rather resulted in impaired efferocytosis by AM, which is necessary for effective resolution of airway inflammation.ConclusionOur findings suggest that NRP2 is a negative regulator of airway inflammation associated with neutrophilic asthma.

Abstract LBA019: The immune checkpoint protein BTN1A1 suppresses T cell activation through interactions with Gal9 and PD-1

Abstract Butyrophilin (BTN) proteins are members of the B7 immunoglobulin superfamily and exhibit well-characterized immunomodulatory functions in mammals. We have recently identified BTN1A1 as an immune checkpoint protein prominently upregulated in response to acute inflammatory insults. Further in vitro and in vivo assays have validated BTN1A1 as an immune checkpoint target, particularly for patients refractory to anti-PD-1/PD-L1 antibody treatment. We have also developed a humanized antibody targeting human BTN1A1, hSTC810, which is expected to enter into Phase I clinical trials in the first quarter of 2022. In this study, a cell microarray from Retrogenix (Whaley Bridge, UK) was used to identify binding partner(s) for the extracellular domain of human BTN1A1. Through this screening approach, we found that BTN1A1 binds to galectin-1 (Gal1), galectin-9 (Gal9), and neuropilin 2 (NRP2). These three putative binding partners could specifically bind to wild-type BTN1A1 but not to this protein's unglycosylated (2NQ) form. Of these three targets, immunoprecipitation and Biacore binding assays revealed that Gal9 exhibited the greatest affinity for human BTN1A1, followed by Gal1, with respective KD values of 22.7 nM and 1.88 μM - an 83-fold difference. Gal9 binding to human BTN1A1 was dependent on BTN1A1 glycosylation status and required the carbohydrate recognition domain (CRD) of Gal9. As Gal9 is a known PD-1-binding protein, the KD of Gal9 for PD-1 was additionally assessed and found to be 19.7 nM. These results thus predicted the potential formation of BTN1A1/Gal9/PD-1 complexes. Consistent with these predictions, immunoprecipitation assays performed using cells expressing Myc-tagged versions of these three proteins demonstrated the formation of BTN1A1/Gal9, PD-1/Gal9, and BTN1A1/Gal9/PD-1 complexes. CRISPR-mediated BTN1A1 knockout in Jurkat T cells induced both PD-1 expression and T cell activation. BTN1A1 also suppressed T cell receptor (TCR) signaling in Jurkat cells, and the addition of exogenous recombinant Gal9 protein further blunted such BTN1A1-mediated TCR-signaling downregulation. Such downregulation was not observed in PD-1 knockout Jurkat cells. Together with the observation that BTN1A1 does not bind to PD-1 directly, the data suggest that BTN1A1 suppresses T cell activation by interacting with PD-1 through Gal9. As high Gal9 expression levels are correlated with poor prognosis in multiple cancers, our results highlight this BTN1A1-Gal9-PD-1 axis as a novel therapeutic target for immunotherapeutic drug development. (1) Chung EM, Bong YS, Kim YS, Park A, You YO, Sharma A, Lin SH, Lee YJ, Jung H, Yoo SS. BTN1A1: a novel immune checkpoint for cancer immunotherapy beyond the PD-1/PD-L1 axis. Cancer Res 2021;81(13_Suppl): Abstract nr 1643. Citation Format: Ezra M Chung, Young-Seung Kim, Chunai Wu, Andrew H Park, Hyunjin Jung, Stephen S Yoo. The immune checkpoint protein BTN1A1 suppresses T cell activation through interactions with Gal9 and PD-1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA019.

Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

Ionizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 – mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell – specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A – NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A – NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo.

Preliminary Study of New Gallium-68 Radiolabeled Peptide Targeting NRP-1 to Detect Brain Metastases by Positron Emission Tomography.

Due to their very poor prognosis and a fatal outcome, secondary brain tumors are one of the biggest challenges in oncology today. From the point of view of the early diagnosis of these brain micro- and macro-tumors, the sensitivity and specificity of the diagnostic tools constitute an obstacle. Molecular imaging, such as Positron Emission Tomography (PET), is a promising technique but remains limited in the search for cerebral localizations, given the commercially available radiotracers. Indeed, the [18F]FDG PET remains constrained by the physiological fixation of the cerebral cortex, which hinders the visualization of cerebral metastases. Tumor angiogenesis is recognized as a crucial phenomenon in the progression of malignant tumors and is correlated with overexpression of the neuropilin-1 (NRP-1) receptor. Here, we describe the synthesis and the photophysical properties of the new gallium-68 radiolabeled peptide to target NRP-1. The KDKPPR peptide was coupled with gallium-68 anchored into a bifunctional NODAGA chelating agent, as well as Cy5 for fluorescence detection. The Cy5 absorbance spectra did not change, whereas the molar extinction coefficient (ε) decreased drastically. An enhancement of the fluorescence quantum yield (φF) could be observed due to the better water solubility of Cy5. [68Ga]Ga-NODAGA-K(Cy5)DKPPR was radiosynthesized efficiently, presented hydrophilic properties (log D = −1.86), and had high in vitro stability (>120 min). The molecular affinity and the cytotoxicity of this new chelated radiotracer were evaluated in vitro on endothelial cells (HUVEC) and MDA-MB-231 cancer cells (hormone-independent and triple-negative line) and in vivo on a brain model of metastasis in a nude rat using the MDA-MB-231 cell line. No in vitro toxicity has been observed. The in vivo preliminary experiments showed promising results, with a high contrast between the healthy brain and metastatic foci for [68Ga]Ga-NODAGA-K(Cy5)DKPPR.

Semaphorin 3A mitigates lipopolysaccharide-induced chondrocyte inflammation, apoptosis and extracellular matrix degradation by binding to Neuropilin-1

ABSTRACT Semaphorin 3A (SEMA3A) and its receptor neuropilin-1 (NRP-1) are expressed low in chondrocytes under stress, and overexpressing SEMA3A reduces pro-inflammatory cytokine release. This study was aimed at exploring whether SEMA3A participates in lipopolysaccharide (LPS)-induced chondrocyte inflammation, apoptosis and extracellular matrix (ECM) degradation. SEMA3A and NRP-1 expression in LPS-induced ATDC5 cells was determined with RT-qPCR and western blotting. Following stimulation with LPS in the absence or presence of SEMA3A overexpression, the viability of ATDC5 cells was observed through CCK-8 assay. RT-qPCR and western blot were performed to detect the expression of pro-inflammatory cytokines. ATDC5 cell apoptosis was observed through TUNEL, and apoptosis-related proteins were assayed. Expression of ECM-related proteins was measured by RT-qPCR and western blotting. Additionally, the binding of SEMA3A to NRP-1 was verified by co-immunoprecipitation. After interference with NRP-1, cell viability, inflammation and ECM degradation were examined in LPS-induced ATDC5 cells with SEMA3A overexpression. Results revealed that SEMA3A expression in ATDC5 cells decreased following stimulation with LPS. Overexpressing SEMA3A improved cell viability and reduced the inflammatory injury of LPS-stimulated ATDC5 cells. Moreover, SEMA3A overexpression alleviated LPS-induced apoptosis and ECM degradation of ATDC5 chondrocytes. SEMA3A and NRP-1 bound to each other in ATDC5 cells. NRP-1 interference crippled the ameliorative effect of SEMA3A overexpression on LPS-induced chondrocyte inflammation, apoptosis and ECM degradation. To conclude, SEMA3A binds to NRP-1, mitigating LPS-induced chondrocyte inflammation, apoptosis and ECM degradation. This study elucidated the role of SEMA3A in osteoarthritis and illustrated its action mechanism involving NRP-1.

Hydroxychloroquine treatment on SARS-CoV-2 receptor ACE2, TMPRSS2 and NRP1 expression in human primary pterygium and conjunctival cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen for coronavirus disease 2019 (COVID-19) pandemic. Its infection depends on the binding of spike protein to the host cell receptor angiotensin-converting enzyme 2 (ACE2), type II transmembrane serine protease (TMPRSS2) and neuropilin-1 (NRP1). Hydroxychloroquine has been applied as one of the COVID-19 treatment strategies. Here we aimed to evaluate hydroxychloroquine treatment on SARS-CoV-2 receptor expression in human primary pterygium and conjunctival cells and its potential influences. Expression of ACE2, TMPRSS2 and NRP1 proteins were found in the epithelial layer of both primary pterygium and conjunctiva tissues as well as in their isolated fibroblasts. High concentration of hydroxychloroquine treatment significantly reduced the viability of both primary pterygium and conjunctival cells. ACE2 protein expression was significantly decreased in both pterygium and conjunctival cells after hydroxychloroquine treatment. Hydroxychloroquine also reduced NRP1 protein expression in conjunctival cells. In contrast, TMPRSS2 protein expression showed slightly increased in conjunctival cells. Notably, ROS production and SOD2 expression was significantly elevated in both pterygium and conjunctival cells after hydroxychloroquine treatment. In summary, this study revealed the reduction of ACE2 and NRP1 expression by hydroxychloroquine in human primary pterygium and conjunctival fibroblasts; yet with the increase in TMPRSS2 expression and oxidative stress and decrease in cell viability. Implementation of hydroxychloroquine for COVID-19 treatment should be carefully considered with its potential side effects and in combination with TMPRSS2 inhibitor.

Sex-Specific Microglial Activation and SARS-CoV-2 Receptor Expression Induced by Chronic Unpredictable Stress.

The coronavirus disease 2019 (COVID-19) pandemic has generated a lot of stress and anxiety among not only infected patients but also the general population across the globe, which disturbs cerebral immune homeostasis and potentially exacerbates the SARS-CoV-2 virus-induced neuroinflammation, especially among people susceptible to neuropsychiatric disorders. Here, we used a chronic unpredictable mild stress (CUMS) mouse model to study its effects on glia-mediated neuroinflammation and expression of SARS-CoV2 viral receptors. We observed that female mice showed depressive-like behavior after CUMS, whereas male mice showed enhanced anxiety and social withdrawal. Interestingly, CUMS led to increased amounts of total and MHCII+ microglia in the hippocampi of female mice but not male mice. mRNA levels of SARS-CoV-2 viral receptors angiotensin-converting enzyme 2 (Ace2) and basigin (Bsg) were also upregulated in the prefrontal cortices of stressed female mice but not male mice. Similarly, sex-specific changes in SARS-CoV-2 viral receptors FURIN and neuropilin-1 (NRP1) were also observed in monocytes of human caregivers enduring chronic stress. Our findings provided evidence on detrimental effects of chronic stress on the brain and behavior and implied potential sex-dependent susceptibility to SARS-CoV-2 infection after chronic stress.

A widespread viral entry mechanism: The C-end Rule motif–neuropilin receptor interaction

Many phylogenetically distant animal viruses, including the new coronavirus severe acute respiratory syndrome coronavirus 2, have surface proteins with polybasic sites that are cleaved by host furin and furin-like proteases. Other than priming certain viral surface proteins for fusion, cleavage generates a carboxy-terminal RXXR sequence. This C-end Rule (CendR) motif is known to bind to neuropilin (NRP) receptors on the cell surface. NRPs are ubiquitously expressed, pleiotropic cell surface receptors with important roles in growth factor signaling, vascular biology, and neurobiology, as well as immune homeostasis and activation. The CendR–NRP receptor interaction promotes endocytic internalization and tissue spreading of different cargo, including viral particles. We propose that the interaction between viral surface proteins and NRPs plays an underappreciated and prevalent role in the transmission and pathogenesis of diverse viruses and represents a promising broad-spectrum antiviral target.

Estimating the viral loads of SARS-CoV-2 in the oral cavity when complicated with periapical lesions

BackgroundThe oral cavity represents a main entrance of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Angiotensin-converting enzyme 2 (ACE-2), neuropilin-1 (NRP-1), and transmembrane serine protease 2 (TMPRSS2) are essential for the entry of SARS-CoV-2 to the host cells. Both ACE-2 and NRP-1 receptors and TMPRSS2 have been identified in the oral cavity. However, there is limited knowledge about the impact of periapical lesions and their metabolites on the expression of these critical genes. This study aims to measure the impact of periapical lesions and their unique fatty acids (FAs) metabolites on the expression of the aforementioned genes, in addition to interleukin 6 (IL-6) gene and hence SARS-CoV-2 infection loads can be estimated.MethodsGene expression of ACE-2, NRP-1, TMPRSS2, and IL-6 was performed in periapical lesions in comparison to healthy oral cavity. Since FAs are important immunomodulators required for the lipid synthesis essential for receptors synthesis and viral replication, comparative FAs profiling was determined in oral lesions and healthy pulp tissues using gas chromatography–mass spectrometry (GC–MS). The effect of major identified and unique FAs was tested on mammalian cells known to express ACE-2, NRP-1, and TMPRSS2 genes.ResultsGene expression analysis indicated that ACE-2, NRP-1, and TMPRSS2 were significantly upregulated in healthy clinical samples compared to oral lesions, while the reverse was true with IL-6 gene expression. Saturated and monounsaturated FAs were the major identified shared and unique FAs, respectively. Major shared FAs included palmitic, stearic and myristic acids with the highest percentage in the healthy oral cavity, while unique FAs included 17-octadecynoic acid in periapical abscess, petroselinic acid and l-lactic acid in periapical granuloma, and 1-nonadecene in the radicular cyst. Computational prediction showed that the binding affinity of identified FAs to ACE-2, TMPRSS2 and S protein were insignificant. Further, FA-treated mammalian cells showed significant overexpression of ACE-2, NRP-1 and TMPRSS2 genes except with l-lactic acid and oleic acid caused downregulation of NRP-1 gene, while 17-octadecynoic acid caused insignificant effect.ConclusionCollectively, a healthy oral cavity is more susceptible to viral infection when compared to that complicated with periapical lesions. FAs play important role in viral infection and their balance can affect the viral loads. Shifting the balance towards higher levels of palmitic, stearic and 1-nonadecene caused significant upregulation of the aforementioned genes and hence higher viral loads. On the other hand, there is a reverse correlation between inflammation and expression of SARS-CoV-2 receptors. Therefore, a mouth preparation that can reduce the levels of palmitic, stearic and 1-nonadecene, while maintaining an immunomodulatory effect can be employed as a future protection strategy against viral infection.

The CBFβ-SMMHC/NRP1 Axis Regulates FLT3 and TGF-Beta Pathways in Inv(16) Acute Myeloid Leukemia

Signal transduction pathways regulate the proliferation and viability of acute myeloid leukemia (AML) blasts. The regulation in the expression of cytokine receptors in AML is not well understood. In this study, we investigated how the CBFβ-SMMHC fusion protein regulates expression of cytokine receptors in inv(16) AML, with focus on the co-receptor Neuropilin-1 (NRP1).Knock-down of CBFβ-SMMHC expression, utilizing shRNA transduction, induced G1 phase of cell cycle arrest and reduced the viability of inv(16) ME-1 cells in culture. Expression profile analysis of CBFβ-SMMHC knock-down cells revealed a significant repression of genes associated with transmembrane receptor protein kinase pathways, including NRP1 (-5 fold), FGFR1 (-4.2 fold) , FLT3 (-2 fold) and TGFBR2 (-1.2 fold). The expression of NRP1 was significantly upregulated in inv(16) AML cases when compared to other AML sub-types and to hematopoietic stem and progenitor cells. Functionally, NRP1 knock-down reduced the viability of ME-1 cells with a similar dynamics as when using CBFβ-SMMHC shRNAs. In addition, the proliferation of inv(16) AML cells was reduced 4.1-fold when treated with a function-blocking antibody for the FV/VIII extracellular NRP1 domain, while having no effect in non-inv(16) AML cells or when using blocking antibody for the CUB extracellular domain. Furthermore, deletion of Nrp1 by gene editing reduced the colony-forming unit capacity of primary mouse Cbfb +/MYH11 leukemic cells and extended the median leukemia latency in vivo. To identify the genes regulated by NRP1 in inv(16) AML, we analysed the transcription profile of NRP1 knock-down in ME-1 cells. Gene Set Enrichment and Pathway Analysis revealed a repression in STAT5 pathway, and in signalling receptor activity, including FLT3 (-1.8 fold) and TGFBR2 (-1.8 fold) expression, indicating that NRP1 mediates transcriptional regulation of FLT3 and TGFBR2 expression in inv(16) AML. Furthermore, the regulation of FLT3 and TGFB2 expression by CBFβ-SMMHC and by NRP1 was validated by gene editing in inv(16) AML blasts. Accordingly, NRP1 knock-down in AML cells reduced SMAD2/3 phosphorylation. The repression of RUNX1/CBFβ function, using small molecule inhibitors, in inv(16) AML cells with CBFβ-SMMHC knockdown restored NRP1 expression, suggesting that RUNX1 may repress NRP1 expression in AML cells. To evaluate if RUNX1 directly regulates NRP1 expression, we tested RUNX1 binding in the NRP1 locus of AML cells with CBFβ-SMMHC knockdown. RUNX1 binding at one of six sites with RUNX1 occupancy identified by chromatin immunoprecipitation followed by sequencing (RE5, regulatory element 5) was increased in the CBFβ-SMMHC knock-down cells. The RE5 is located 178kb upstream of the NRP1 transcription start site and it is evolutionarily conserved in vertebrates. The deletion of RE5 by gene editing (~50% editing efficiency) increased NRP1 expression 1.8-fold, suggesting that RUNX1 may repress NRP1 expression at by binding to the RE5 enhancer.Taken together, these studies demonstrate that CBFβ-SMMHC regulates expression of cytokine receptors in inv(16) AML. Specifically, it directly regulates expression of the co-receptor NRP1, which is essential for AML survival, acting (at least in part) by regulating FLT3 and TGFB pathways. DisclosuresGuzman: SeqRx: Consultancy; BridgeMedicines: Consultancy; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1.

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF‐B growth factor is involved in cell survival, anti‐apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin‐1 (NRP1). We employed surface plasmon resonance technology and X‐ray crystallography to analyse the molecular basis of the interaction between VEGF‐B and the b1 domain of NRP1, and developed VEGF‐B C‐terminus derived peptides to be used as chemical tools for studying VEGF‐B ‐ NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF‐B‐derived peptides containing a C‐terminal arginine show potent binding to NRP1‐b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF‐B peptides bind at the canonical C‐terminal arginine binding site. VEGF‐B C‐terminus imparts higher affinity for NRP1 than the corresponding VEGF‐A165 region. This tight binding may impact on the activity and selectivity of the full‐length protein. The VEGF‐B167 derived peptides were more effective than VEGF‐A165 peptides in blocking functional phosphorylation events. Blockers of VEGF‐B function have potential applications in diabetes and non‐alcoholic fatty liver disease.

Maternal plasma soluble neuropilin-1 is downregulated in fetal growth restriction complicated by abnormal umbilical artery Doppler: a pilot study.

ABSTRACTObjectivesPlacental expression of neuropilin‐1 (NRP1), a proangiogenic member of the vascular endothelial growth factor receptor family involved in sprouting angiogenesis, was recently discovered to be downregulated in pregnancies with fetal growth restriction (FGR) and abnormal umbilical artery (UA) Doppler. Soluble NRP1 (sNRP1) is an antagonist to NRP1; however, little is known about its role in normal and FGR pregnancies. This study tested the hypotheses that, first, sNRP1 would be detectable in maternal circulation and, second, its concentration would be upregulated in FGR pregnancies compared to those with normal fetal growth and this would correlate with the severity of the disease as assessed by UA Doppler.MethodsThis was a prospective case–control pilot study of 40 singleton pregnancies (20 FGR cases and 20 uncomplicated controls) between 24 + 0 and 40 + 0 weeks' gestation followed in an academic perinatal center from January 2015 to May 2017. FGR was defined as an ultrasound‐estimated fetal weight < 10th percentile for gestational age. The control group was matched to the FGR group for maternal age and gestational age at assessment. Fetal ultrasound biometry and UA Doppler were performed using standard protocols. Maternal plasma sNRP1 measurements were performed using a commercially available ELISA.ResultsContrary to the study hypothesis, maternal plasma sNRP1 levels were significantly decreased in FGR pregnancies as compared to those with normal fetal growth (137.4 ± 44.8 pg/mL vs 166.7 ± 36.9 pg/mL; P = 0.03). However, there was no significant difference in sNRP1 concentration between the control group and FGR pregnancies that had normal UA Doppler. Plasma sNRP1 was downregulated in FGR pregnancies with elevated UA systolic/diastolic ratio (P = 0.023) and those with UA absent or reversed end‐diastolic flow (P = 0.005) in comparison to FGR pregnancies with normal UA Doppler. This suggests that biometrically small fetuses without hemodynamic compromise are small‐for‐gestational age rather than FGR.ConclusionsThis study demonstrated a significant decrease in maternal plasma sNRP1 concentration in growth‐restricted pregnancies with fetoplacental circulatory compromise. These findings suggest a possible role of sNRP1 in modulating fetal growth and its potential as a biomarker for FGR. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Microfluidic preparation and in vitro evaluation of iRGD-functionalized solid lipid nanoparticles for targeted delivery of paclitaxel to tumor cells

Solid lipid nanoparticles (SLNs) can combine the advantages of different colloidal carriers and prevent some of their disadvantages. The production of nanoparticles by means of microfluidics represents a successful platform for industrial scale-up of nanoparticle manufacture in a reproducible way. The realisation of a microfluidic technique to obtain SLNs in a continuous and reproducible manner encouraged us to create surface functionalised SLNs for targeted drug release using the same procedure. A tumor homing peptide, iRGD, owning a cryptic C-end Rule (CendR) motif is responsible for neuropilin-1 (NRP-1) binding and for triggering extravasation and tumor penetration of the peptide. In this study, the Paclitaxel loaded-SLNs produced by microfluidics were functionalized with the iRGD peptide. The SLNs proved to be stable in aqueous medium andwere characterized by a Z-average under 150 nm, a polydispersity index below 0.2, a zeta-potential between −20 and −35 mV and a drug encapsulation efficiency around 40%. Moreover, in vitro cytotoxic effects and cellular uptake have been assessed using 2D and 3D tumour models of U87 glioblastoma cell lines. Overall, these results demonstrate that the surface functionalization of SLNs with iRGD allow better cellular uptake and cytotoxicity ability.

Cardiac Fibrosis Is a Risk Factor for Severe COVID-19.

Increased left ventricular fibrosis has been reported in patients hospitalized with coronavirus disease 2019 (COVID-19). It is unclear whether this fibrosis is a consequence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection or a risk factor for severe disease progression. We observed increased fibrosis in the left ventricular myocardium of deceased COVID-19 patients, compared with matched controls. We also detected increased mRNA levels of soluble interleukin-1 receptor-like 1 (sIL1-RL1) and transforming growth factor β1 (TGF-β1) in the left ventricular myocardium of deceased COVID-19 patients. Biochemical analysis of blood sampled from patients admitted to the emergency department (ED) with COVID-19 revealed highly elevated levels of TGF-β1 mRNA in these patients compared to controls. Left ventricular strain measured by echocardiography as a marker of pre-existing cardiac fibrosis correlated strongly with blood TGF-β1 mRNA levels and predicted disease severity in COVID-19 patients. In the left ventricular myocardium and lungs of COVID-19 patients, we found increased neuropilin-1 (NRP-1) RNA levels, which correlated strongly with the prevalence of pulmonary SARS-CoV-2 nucleocapsid. Cardiac and pulmonary fibrosis may therefore predispose these patients to increased cellular viral entry in the lung, which may explain the worse clinical outcome observed in our cohort. Our study demonstrates that patients at risk of clinical deterioration can be identified early by echocardiographic strain analysis and quantification of blood TGF-β1 mRNA performed at the time of first medical contact.

Identifying New COVID-19 Receptor Neuropilin-1 in Severe Alzheimer's Disease Patients Group Brain Using Genome-Wide Association Study Approach.

Recent preclinical studies show that Neuropilin-1 (NRP1), which is a transmembrane protein with roles in neuronal development, axonal outgrowth, and angiogenesis, also plays a role in the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Thus, we hypothesize that NRP1 may be upregulated in Alzheimer’s disease (AD) patients and that a correlation between AD and SARS-CoV-2 NRP1-mediated infectivity may exist as angiotensin converting enzyme 2 (ACE2). We used an AD mouse model that mimics AD and performed high-throughput total RNA-seq with brain tissue and whole blood. For quantification of NRP1 in AD, brain tissues and blood were subjected to Western blotting and real-time quantitative PCR (RT-qPCR) analysis. In silico analysis for NRP1 expression in AD patients has been performed on human hippocampus data sets. Many cases of severe symptoms of COVID-19 are concentrated in an elderly group with complications such as diabetes, degenerative disease, and brain disorders. Total RNA-seq analysis showed that the Nrp1 gene was commonly overexpressed in the AD model. Similar to ACE2, the NRP1 protein is also strongly expressed in AD brain tissues. Interestingly, in silico analysis revealed that the level of expression for NRP1 was distinct at age and AD progression. Given that NRP1 is highly expressed in AD, it is important to understand and predict that NRP1 may be a risk factor for SARS-CoV-2 infection in AD patients. This supports the development of potential therapeutic drugs to reduce SARS-CoV-2 transmission.