Neuropilins as potential biomarkers in hepatocellular carcinoma: a systematic review of basic and clinical implications.

Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers worldwide and is characterized by complex molecular carcinogenesis. Neuropilins (NRPs) NRP1 and NRP2 are the receptors of multiple proteins involved in key signaling pathways associated with tumor progression. We aimed to systematically review all the available findings on their role in HCC. We searched the Scopus, Web of Science (WOS), PubMed, Cochrane and Embase databases for articles evaluating NRPs in preclinical or clinical HCC models. This study was registered in PROSPERO (CRD42022349774) and include 49 studies. Multiple cellular and molecular processes have been associated with one or both NRPs, indicating that they are potential diagnostic and prognostic biomarkers in HCC patients. Mainly NRP1 has been shown to promote tumor cell survival and progression by modulating several signaling pathways. NRPs mainly regulate angiogenesis, invasion and migration and have shown to induce invasion and metastasis. They also regulate the immune response and tumor microenvironment, showing a crucial interplay with the hypoxia response and microRNAs in HCC. Altogether, NRP1 and NRP2 are potential biomarkers and therapeutic targets, providing novel insight into the clinical landscape of HCC patients.

VEGFA, B, C: Implications of the C-Terminal Sequence Variations for the Interaction with Neuropilins.

Vascular endothelial growth factors (VEGFs) are the key regulators of blood and lymphatic vessels' formation and function. Each of the proteins from the homologous family VEGFA, VEGFB, VEGFC and VEGFD employs a core cysteine-knot structural domain for the specific interaction with one or more of the cognate tyrosine kinase receptors. Additional diversity is exhibited by the involvement of neuropilins-transmembrane co-receptors, whose b1 domain contains the binding site for the C-terminal sequence of VEGFs. Although all relevant isoforms of VEGFs that interact with neuropilins contain the required C-terminal Arg residue, there is selectivity of neuropilins and VEGF receptors for the VEGF proteins, which is reflected in the physiological roles that they mediate. To decipher the contribution made by the C-terminal sequences of the individual VEGF proteins to that functional differentiation, we determined structures of molecular complexes of neuropilins and VEGF-derived peptides and examined binding interactions for all neuropilin-VEGF pairs experimentally and computationally. While X-ray crystal structures and ligand-binding experiments highlighted similarities between the ligands, the molecular dynamics simulations uncovered conformational preferences of VEGF-derived peptides beyond the C-terminal arginine that contribute to the ligand selectivity of neuropilins. The implications for the design of the selective antagonists of neuropilins' functions are discussed.

A Potential Role of Semaphorin 3A during Orthodontic Tooth Movement.

BACKGROUND: Induced tooth movement during orthodontic therapy requires mechano-induced bone remodeling. Besides various cytokines and growth-factors, neuronal guidance molecules gained attention for their roles in bone homeostasis and thus, potential roles during tooth movement. Several neuronal guidance molecules have been implicated in the regulation of bone remodeling. Amongst them, Semaphorin 3A is particular interesting as it concurrently induces osteoblast differentiation and disturbs osteoclast differentiation. METHODS: Mechano-regulation of Sema3A and its receptors PlexinA1 and Neuropilin (RT-qPCR, WB) was evaluated by applying compressive and tension forces to primary human periodontal fibroblasts (hPDLF) and alveolar bone osteoblasts (hOB). The association of the transcription factor Osterix (SP7) and SEMA3A was studied by RT-qPCR. Mechanisms involved in SEMA3A-mediated osteoblast differentiation were assessed by Rac1GTPase pull-downs, ß-catenin expression analyses (RT-qPCR) and nuclear translocation assays (IF). Osteogenic markers were analyzed by RT-qPCR. RESULTS: SEMA3A, PLXNA1 and NRP1 were differentially regulated by tension or compressive forces in hPDLF. Osterix (SP7) displayed the same pattern of regulation. Recombinant Sema3A induced the activation of Rac1GTPase, the nuclear translocation of ß-catenin and the expression of osteogenic marker genes. CONCLUSION: Sema3A, its receptors and Osterix are regulated by mechanical forces in hPDLF. SEMA3A upregulation was associated with Osterix (SP7) modulation. Sema3A-enhanced osteogenic marker gene expression in hOB might be dependent on a pathway involving Rac1GTPase and ß-catenin. Thus, Semaphorin 3A might contribute to bone remodeling during induced tooth movement.

Targeting neuropilins as a viable SARS-CoV-2 treatment.

The SARS-CoV-2 pandemic has significantly impacted global health. Research on viral mechanisms, highly effective vaccines, and other therapies is in progress. Neuropilins have recently been identified as host cell receptors enabling viral fusion. Here, we provide context to neuropilin's tissue-specific role in infection and the potential impact of NRP-based therapeutics. We conclude that the central roles of neuropilins in vascular, neural, and other pathways may render it a less suitable target for treating SARS-CoV-2 than agents that target its binding partner, the viral spike protein.

Pancancer Analysis of Neurovascular-Related NRP Family Genes as Potential Prognostic Biomarkers of Bladder Urothelial Carcinoma.

BACKGROUND: Neurovascular-related genes have been implicated in the development of cancer. Studies have shown that a high expression of neuropilins (NRPs) promotes tumourigenesis and tumour malignancy. METHOD: A multidimensional bioinformatics analysis was performed to examine the relationship between NRP genes and prognostic and pathological features, tumour mutational burden (TMB), microsatellite instability (MSI), and immunological features based on public databases and find the potential prognostic value of NRPs in pancancer. RESULTS: Survival analysis revealed that a low NRP1 expression in adrenocortical carcinoma (ACC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), low-grade glioma (LGG), and stomach adenocarcinoma (STAD) was associated with poor prognosis. A high NRP2 expression in bladder urothelial carcinoma (BLCA), kidney renal papillary cell carcinoma (KIRP), and mesothelioma (MESO) was associated with poor prognosis. Moreover, NRP1 and NRP2 were associated with TMB and MSI. Subsequent analyses showed that NRP1 and NRP2 were correlated with immune infiltration and immune checkpoints. Genome-wide association analysis revealed that the NRP1 expression was strongly associated with kidney renal clear cell carcinoma (KIRC), whereas the NRP2 expression was closely associated with BLCA. Ultimately, NRP2 was found to be involved in the development of BLCA. CONCLUSIONS: Neurovascular-related NRP family genes are significantly correlated with cancer prognosis, TME, and immune infiltration, particularly in BLCA.

Semaphorins as emerging clinical biomarkers and therapeutic targets in cancer.

Semaphorins are a large family of developmental regulatory signals, characterized by aberrant expression in human cancers. These molecules crucially control cell-cell communication, cell migration, invasion and metastasis, tumor angiogenesis, inflammatory and anti-cancer immune responses. Semaphorins comprise secreted and cell surface-exposed molecules and their receptors are mainly found in the Plexin and Neuropilin families, which are further implicated in a signaling network controlling the tumor microenvironment. Accumulating evidence indicates that semaphorins may be considered as novel clinical biomarkers for cancer, especially for the prediction of patient survival and responsiveness to therapy. Moreover, preclinical experimental studies have demonstrated that targeting semaphorin signaling can interfere with tumor growth and/or metastatic dissemination, suggesting their relevance as novel therapeutic targets in cancer; this has also prompted the development of semaphorin-interfering molecules for application in the clinic. Here we will survey, in diverse human cancers, the current knowledge about the relevance of semaphorin family members, and conceptualize potential lines of future research development in this field.

Presence of the Transmembrane Protein Neuropilin in Cytokine-induced Killer Cells.

BACKGROUND/AIM: Cytokine-induced killer (CIK) cells are a heterogenous population of immune cells showing promising applications in immunotherapeutic cancer treatment. Neuropilin (NRP) proteins have been proven to play an important role in cancer development and prognosis. In this study, CIK cells were tested for expression of NRPs, transmembrane proteins playing a role in the proliferation and survival of cancer cells. MATERIALS AND METHODS: CIK cells were analyzed at different time points via flow cytometry and quantitative real-time polymerase chain reaction for neuropilin expression. RESULTS: Phenotyping results showed CIK cells having developed properly, and low levels of NRP2 were detected. On the other hand, no NRP1 expression was found. Two cancer cell lines were tested by flow cytometry: A549 cells expressed NRP1 and NRP2; U251-MG cells expressed high amounts of NRP2. CIK cell showed low levels of NRP2 expression on day 14. CONCLUSION: The presence of NRP2, but not NRP1, was shown for CIK cells. Recognizing NRP2 in CIK cells might help to improve CIK cell cytotoxicity.

Semaphorin/neuropilin binding specificities are stable over 400 million years of evolution.

Semaphorins are a large and important family of signaling molecules conserved in Bilateria. An important determinant of the biological function of their largest class, the secreted class 3 semaphorins, is the specificity of their binding to neuropilins, a key component of a larger holoreceptor complex. We compared these binding specificities in mice and zebrafish, species whose most recent common ancestor was more than 400 million years in the past. We also compared the binding specificities of zebrafish class 3 semaphorins that were duplicated very early within the teleost lineage. We found a surprising conservation of neuropilin binding specificities when comparing both paralogous zebrafish semaphorin pairs and orthologous zebrafish and mouse semaphorin pairs. This finding was further supported by a remarkable conservation of binding specificities in cross-species pairings of semaphorins and neuropilins. Our results suggest that the qualitative specificities with which particular semaphorins bind to particular neuropilins has remained nearly invariant over approximately 400 million years of evolution.

The VEGF receptor neuropilin 2 promotes homologous recombination by stimulating YAP/TAZ-mediated Rad51 expression.

Vascular endothelial growth factor (VEGF) signaling in tumor cells mediated by neuropilins (NRPs) contributes to the aggressive nature of several cancers, including triple-negative breast cancer (TNBC), independently of its role in angiogenesis. Understanding the mechanisms by which VEGF-NRP signaling contributes to the phenotype of such cancers is a significant and timely problem. We report that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double-strand break repair. Mechanistically, we provide evidence that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. We also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to cisplatin and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ. These findings reveal roles for VEGF-NRP2 and YAP/TAZ in DNA repair, and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ, and Rad51 that contributes to resistance to platinum chemotherapy.

VEGF/Neuropilin Signaling in Cancer Stem Cells.

The function of vascular endothelial growth factor (VEGF) in cancer extends beyond angiogenesis and vascular permeability. Specifically, VEGF-mediated signaling occurs in tumor cells and this signaling contributes to key aspects of tumorigenesis including the self-renewal and survival of cancer stem cells (CSCs). In addition to VEGF receptor tyrosine kinases, the neuropilins (NRPs) are critical for mediating the effects of VEGF on CSCs, primarily because of their ability to impact the function of growth factor receptors and integrins. VEGF/NRP signaling can regulate the expression and function of key molecules that have been implicated in CSC function including Rho family guanosine triphosphatases (GTPases) and transcription factors. The VEGF/NRP signaling axis is a prime target for therapy because it can confer resistance to standard chemotherapy, which is ineffective against most CSCs. Indeed, several studies have shown that targeting either NRP1 or NRP2 can inhibit tumor initiation and decrease resistance to other therapies.

An ancestral role for Semaphorin3F-Neuropilin signaling in patterning neural crest within the new vertebrate head.

The origin of the vertebrate head is one of the great unresolved issues in vertebrate evolutionary developmental biology. Although many of the novelties in the vertebrate head and pharynx derive from the neural crest, it is still unknown how early vertebrates patterned the neural crest within the ancestral body plan they inherited from invertebrate chordates. Here, using a basal vertebrate, the sea lamprey, we show that homologs of Semaphorin3F (Sema3F) ligand and its Neuropilin (Nrp) receptors show complementary and dynamic patterns of expression that correlate with key periods of neural crest development (migration and patterning of cranial neural crest-derived structures). Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that lamprey Sema3F is essential for patterning of neural crest-derived melanocytes, cranial ganglia and the head skeleton, but is not required for neural crest migration or patterning of trunk neural crest derivatives. Based on comparisons with jawed vertebrates, our results suggest that the deployment of Nrp-Sema3F signaling, along with other intercellular guidance cues, was pivotal in allowing early vertebrates to organize and pattern cranial neural crest cells into many of the hallmark structures that define the vertebrate head.

VEGF-A121a binding to Neuropilins - A concept revisited.

All known splice isoforms of vascular endothelial growth factor A (VEGF-A) can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2. We focus here on VEGF-A121a and VEGF-A165a, two of the most abundant VEGF-A splice isoforms in human tissue 1 , and their ability to bind the Neuropilin co-receptors NRP1 and NRP2. The Neuropilins are key vascular, immune, and nervous system receptors on endothelial cells, neuronal axons, and regulatory T cells respectively. They serve as co-receptors for the Plexins in Semaphorin binding on neuronal and vascular endothelial cells, and for the VEGFRs in VEGF binding on vascular and lymphatic endothelial cells, and thus regulate the initiation and coordination of cell signaling by Semaphorins and VEGFs. 2 There is conflicting evidence in the literature as to whether only heparin-binding VEGF-A isoforms - that is, isoforms with domains encoded by exons 6 and/or 7 plus 8a - bind to Neuropilins on endothelial cells. While it is clear that VEGF-A165a binds to both NRP1 and NRP2, published studies do not all agree on the ability of VEGF-A121a to bind NRPs. Here, we review and attempt to reconcile evidence for and against VEGF-A121a binding to Neuropilins. This evidence suggests that, in vitro, VEGF-A121a can bind to both NRP1 and NRP2 via domains encoded by exons 5 and 8a; in the case of NRP1, VEGF-A121a binds with lower affinity than VEGF-A165a. In in vitro cell culture experiments, both NRP1 and NRP2 can enhance VEGF-A121a-induced phosphorylation of VEGFR2 and downstream signaling including proliferation. However, unlike VEGFA-165a, experiments have shown that VEGF-A121a does not 'bridge' VEGFR2 and NRP1, i.e. it does not bind both receptors simultaneously at their extracellular domain. Thus, the mechanism by which Neuropilins potentiate VEGF-A121a-mediated VEGFR2 signaling may be different from that for VEGF-A165a. We suggest such an alternate mechanism: interactions between NRP1 and VEGFR2 transmembrane (TM) and intracellular (IC) domains.

The semaphorins and their receptors as modulators of tumor progression.

The semaphorins were initially characterized as repulsive axon guidance factors. However, they are currently also recognized as important regulators of diverse biological processes which include regulation of immune responses, angiogenesis, organogenesis, and a variety of additional physiological and developmental functions. The semaphorin family consists of more than 20 genes divided into seven subfamilies, all of which contain the sema domain signature. They usually transduce signals by activation of receptors belonging to the plexin family, either directly, or indirectly following the binding of some semaphorins to receptors of the neuropilin family which subsequently associate with plexins. Additional receptors which form complexes with these primary semaphorin receptors are also frequently involved in semaphorin signalling, and can strongly influence the nature of the biological responses of cells to semaphorins. Recent evidence suggests that semaphorins play important roles in the etiology of multiple forms of cancer. Some semaphorins such as some semaphorins belonging to the class-3 semaphorin subfamily, have been found to function as bona fide tumor suppressors and to inhibit tumor progression by various mechanisms. Because these class-3 semaphorins are secreted proteins, these semaphorins may potentially be used as anti-tumorigenic drugs. Other semaphorins, such as semaphorin-4D, function as inducers of tumor progression and represent targets for the development of novel anti-tumorigenic drugs. The mechanisms by which semaphorins affect tumor progression are diverse, ranging from direct effects on tumor cells to modulation of accessory processes such as modulation of immune responses and inhibition or promotion of tumor angiogenesis and tumor lymphangiogenesis. This review focuses on the diverse mechanisms by which semaphorins affect tumor progression.

The neuropilin-like protein ESDN regulates insulin signaling and sensitivity.

Insulin effects on cell metabolism, growth, and survival are mediated by its binding to, and activation of, insulin receptor. With increasing prevalence of insulin resistance and diabetes there is considerable interest in identifying novel regulators of insulin signal transduction. The transmembrane protein endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a novel regulator of vascular remodeling and angiogenesis. Here, we investigate a potential role of ESDN in insulin signaling, demonstrating that Esdn gene deletion promotes insulin-induced vascular smooth muscle cell proliferation and migration. This is associated with enhanced protein kinase B and mitogen-activated protein kinase activation as well as insulin receptor phosphorylation. Likewise, insulin signaling in the liver, muscle, and adipose tissue is enhanced in Esdn(-/-) mice, and these animals exhibit improved insulin sensitivity and glucose homeostasis in vivo. The effect of ESDN on insulin signaling is traced back to its interaction with insulin receptor, which alters the receptor interaction with regulatory adaptor protein-E3 ubiquitin ligase pairs, adaptor protein with pleckstrin homology and Src homology 2 domain-c-Cbl and growth factor receptor bound protein 10-neuronal precursor cell-expressed developmentally downregulated 4. In conclusion, our findings establish ESDN as an inhibitor of insulin receptor signal transduction through a novel regulatory mechanism. Loss of ESDN potentiates insulin's metabolic and mitotic effects and provides insights into a novel therapeutic avenue.

Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Deficiencies of the human cystathionine ß-synthase (CBS) enzyme are characterized by a plethora of vascular disorders and hyperhomocysteinemia. However, several clinical trials demonstrated that despite reduction in homocysteine levels, disease outcome remained unaffected, thus the mechanism of endothelial dysfunction is poorly defined. Here, we show that the loss of CBS function in endothelial cells (ECs) leads to a significant down-regulation of cellular hydrogen sulfide (H2S) by 50% and of glutathione (GSH) by 40%. Silencing CBS in ECs compromised phenotypic and signaling responses to the VEGF that were potentiated by decreased transcription of VEGF receptor (VEGFR)-2 and neuropilin (NRP)-1, the primary receptors regulating endothelial function. Transcriptional down-regulation of VEGFR-2 and NRP-1 was mediated by a lack in stability of the transcription factor specificity protein 1 (Sp1), which is a sulfhydration target of H2S at residues Cys68 and Cys755. Reinstating H2S but not GSH in CBS-silenced ECs restored Sp1 levels and its binding to the VEGFR-2 promoter and VEGFR-2, NRP-1 expression, VEGF-dependent proliferation, and migration phenotypes. Thus, our study emphasizes the importance of CBS-mediated protein S-sulfhydration in maintaining vascular health and function.-Saha, S., Chakraborty, P. K., Xiong, X., Dwivedi, S. K. D., Mustafi, S. B., Leigh, N. R., Ramchandran, R., Mukherjee, P., Bhattacharya, R. Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Semaphorins and neuropilins expression in salivary gland tumors.

BACKGROUND: Salivary gland tumors (SGT) account for 3-10% of all head and neck neoplasms, and little is known about their angiogenic properties. Despite semaphorins and neuropilins have been demonstrated to be prognostic determinants in many human cancers, they remain to be investigated in SGT. Therefore, the objective of this study was to analyze the clinical significance of the expression of class 3 semaphorins A (Sema3A) and B (Sema3B) and neuropilins-1 (Np-1) and neuropilins-2 (Np-2), in SGT. METHODS: Two hundred and forty-eight SGT were organized in tissue microarray paraffin blocks and expression of CD34, Sema3A, Sema3B, Np-1, and Np-2 was determined through immunohistochemistry. The immunoreactions were quantified using digital algorithms and the results correlated with clinicopathological parameters. RESULTS: Malignant tumors had an increased vascular density than their benign counterparts and their increased vascular area significantly correlated with recurrences (P < 0.05). Patients older than 40 years and the presence of recurrences determined an inferior survival rate (P = 0.0057 and P = 0.0303, respectively). In normal salivary glands, Np-1 and Np-2 expression was restricted to ductal cells, whereas Sema3A and Sema3B were positive in the serous acinar compartment. Tumors were positive for all markers and the co-expression of Np-1/Np-2 significantly correlated with the presence of paresthesia and advanced stages of the tumors (P = 0.01 and P = 0.04, respectively). CONCLUSION: Sema3A, Sema3B, Np-1, and Np-2 may be involved in the pathogenesis of SGT, but their expression did not present a statistically significant prognostic potential in this study.

Changes in expression of Class 3 Semaphorins and their receptors during development of the rat retina and superior colliculus.

BACKGROUND: Members of the Semaphorin 3 family (Sema3s) influence the development of the central nervous system, and some are implicated in regulating aspects of visual system development. However, we lack information about the timing of expression of the Sema3s with respect to different developmental epochs in the mammalian visual system. In this time-course study in the rat, we document for the first time changes in the expression of RNAs for the majority of Class 3 Semaphorins (Sema3s) and their receptor components during the development of the rat retina and superior colliculus (SC). RESULTS: During retinal development, transcript levels changed for all of the Sema3s examined, as well as Nrp2, Plxna2, Plxna3, and Plxna4a. In the SC there were also changes in transcript levels for all Sema3s tested, as well as Nrp1, Nrp2, Plxna1, Plxna2, Plxna3, and Plxna4a. These changes correlate with well-established epochs, and our data suggest that the Sema3s could influence retinal ganglion cell (RGC) apoptosis, patterning and connectivity in the maturing retina and SC, and perhaps guidance of RGC and cortical axons in the SC. Functionally we found that SEMA3A, SEMA3C, SEMA3E, and SEMA3F proteins collapsed purified postnatal day 1 RGC growth cones in vitro. Significantly this is a developmental stage when RGCs are growing into and within the SC and are exposed to Sema3 ligands. CONCLUSION: These new data describing the overall temporal regulation of Sema3 expression in the rat retina and SC provide a platform for further work characterising the functional impact of these proteins on the development and maturation of mammalian visual pathways.

miR-1247 is correlated with prognosis of pancreatic cancer and inhibits cell proliferation by targeting neuropilins.

Accumulating evidence indicates that microRNAs (miRNAs) have great potential as tumor biomarkers and therapeutic agents owing to their functions in tumorigenesis and cancer progression. Aberrant expression of miR-1247 has been found in several cancers and is predicted to play an important role in the pathological processes of pancreatic cancer by miRNA-regulated network analysis. We investigated the expression profile of miR-1247 in pancreatic cancer tissue microarray by in situ hybridization and found that miR-1247 was significantly down-regulated in pancreatic cancer tissues compared to matched benign tissues. High levels of miR-1247 expression were positively correlated with higher overall and recurrence free survival in pancreatic cancer patients, while negatively correlated with tumor grade. Using in vitro and in vivo models, we demonstrated that increased expression of miR-1247 inhibited proliferation, tumorigenicity, colony formation and triggered G0/G1 cell cycle arrest in pancreatic cancer cells. Moreover, we confirmed that neuropilin1 (NRP1) and neuropilin2 (NRP2) are direct targets of miR-1247 by western blot and luciferase reporter assay. Further studies indicated that low dose all trans retinoic acid (ATRA) can induce redifferentiation and restoration of miR-1247 in pancreatic cancer cells. These findings suggest that miR-1247, a novel tumor suppressor, can act as a potential biomarker and therapeutic agent for pancreatic cancer.

Constitutive expression of vascular endothelial cell growth factor (VEGF) gene family ligand and receptors on human upper and lower airway epithelial cells.

BACKGROUND: We previously reported that vascular endothelial cell growth factor (VEGF) is abundantly expressed by primary human nasal epithelial cells (PNECs) and functions to promote cell hyperplasia in polyposis. Therefore, we aimed to examine the full expression profile of other members of the VEGF gene family of ligands and receptors, which may play a role in cell growth and the development of chronic rhinosinusitis with nasal polyposis (CRSwNP). METHODS: Messenger RNA (mRNA) and protein expression of VEGF genes, receptors, and co-receptors was examined from cultured PNECs (n = 4) and compared to that from primary human bronchial epithelial cells (PBECs; n = 4) and the BEAS2B cell line (n = 4) by real-time polymerase chain reaction (PCR) and flow cytometry. RESULTS: We report abundant expression of VEGFA, VEGFB, and VEGFC, detected by mRNA and flow cytometric analysis on PNECs. We herein report the novel finding that there is significant expression of VEGFR1, VEGFR2, VEGFR3, and both neuropilin co-receptors, NP1 and NP2, at baseline conditions on PNECs. Lower airway PBECs and BEAS2B cells displayed similar patterns of expression. CONCLUSION: PNECs express high constitutive levels of the VEGF gene family homolog of ligands and receptors. Expression of multiple VEGF ligand-receptor combinations may function as redundant pathways to promote upper and lower airway epithelial cell growth during inflammation.

Enhancement of the tumor penetration of monoclonal antibody by fusion of a neuropilin-targeting peptide improves the antitumor efficacy.

The limited localization and penetration of monoclonal antibodies (mAb) into solid tumors restricts their antitumor efficacy. Here, we describe a solid tumor-targeting antibody with enhanced tumor penetration activity. We designed a 22-residue peptide (A22p), which was extracted from the C-terminal basic region of semaphorin 3A (Sema3A) but modified to have higher affinity with neuropilin receptors (NRP), and genetically fused it to the C-terminus of Fc of human immunoglobulin G1 via a 15-residue (G4S)3 linker, generating Fc-A22p, for the bivalent binding to NRPs. In contrast to Fc or the monovalent A22p peptide alone, Fc-A22p homed to tumor vessels and induced vascular permeability through VE-cadherin downregulation and penetrated tumor tissues by interacting with NRPs in mice bearing human tumor xenografts. We extended the Fc-A22p platform by generating mAb-A22p antibodies of two clinically approved solid tumor-targeting mAbs, the anti-EGF receptor mAb cetuximab (erbitux), and the anti-Her2 mAb trastuzumab (herceptin). The mAb-A22p antibodies retained the intrinsic antigen binding, natural Fc-like biophysical properties, and productivity in mammalian cell cultures, comparable with those of the parent mAbs. In mouse xenograft tumor models, the mAb-A22p antibodies more efficiently homed to tumor vessels and spread into the extravascular tumor parenchyma, which significantly enhanced antitumor efficacy compared with the parent mAbs. Our results suggest that mAb-A22p is a superior format for solid tumor-targeting antibodies due to its enhanced tumor tissue penetration and greater antitumor efficacy compared with conventional mAbs.