Netrin-4: Focus on Its Role in Axon Guidance, Tissue Stability, Angiogenesis and Tumors

Netrin-4, a member of the Netrins family, is an important secreted protein that plays a role in axonal outgrowth and migration orientation. It was initially described that Netrin-4 had a high correlation with the laminin β-chain and promoted the growth of neurites in cultured olfactory bulb explants. Subsequently, it was discovered that Netrin-4 is involved in regulating various physiological processes, including angiogenesis, the occurrence and metastasis of various tumors, and the development of the kidney and alveoli. This paper reviews the current research on Netrin-4 since its discovery and provides a theoretical basis for further research on the biological characteristics of Netrin-4. Graphical Abstract Effects of Netrin-4. Netrin-4 regulates axon guidance, angiogenesis and the development of various tumors.

Netrin-4, Axon guidance, Angiogenesis, Tumor, DCC, Unc5, Neogenin

Netrin-4, also called β-netrin, is a member of the Netrins family, which is a laminin-like secreted protein of regulators of axonal guidance. It is vitally important to the development of the central nervous system, bone, kidney and blood vessels (Koch et al. 2000; Zhang et al. 2004; Qin et al. 2007; Hayano et al. 2014; Enoki et al. 2017), as well as in the development and metastasis of various cancers (Hu et al. 2012; Srivastava et al. 2015; Yuan et al. 2011). There are four families of axon guidance factors, including netrins, ephrin, slit and semaphorins. Netrins belong to a family of axon guidance factors with highly conserved structures. To date, five autocrine and paracrine proteins, Netrin-1–5 (Garrett et al. 2016), and two membrane-binding proteins, Netrin-G1 and Netrin-G2, have been authenticated (Larrieu-Lahargue et al. 2012). In 1990, the Netrins family Unc6 was first discovered in Caenorhabditis elegans and demonstrated to be involved in cell migration and axon guidance (Bradford et al. 2009). In 1994, Netrin-1, a homolog of Unc6, was discovered in vertebrates (Serafini et al. 1994). The human Netrin-1 gene is located in 17p13.1, which encodes 604 amino acids and is approximately 67.7 kDa. Netrin-2 is expressed in birds and zebrafish, while Netrin-3 is a homologous gene of Netrin-2 in mammals, located at 16p13.3, encoding 580 amino acids and approximately 61 kDa. Human Netrin-4 (Koch et al. 2000) is homologous to the NH2 terminus of the short arm of laminin. The gene is located in 12q22.q23 and encodes 628 amino acids approximately 70–84 kDa. However, Netrin-5, a member of the family discovered in 2015, is different from other members: Netrin-5a and Netrin-5b lack an EGF-like sequence of epidermal growth factor (EGF) in the laminin VI domain and the laminin V domain. Netrin-5b is missing the C345C domain, and Netrin-5c is smaller than Netrin-5a but consists of three domains similar to EGF (Yamagishi et al. 2015). Netrin-G, which was described by Nakashiba and coworkers, is characterized predominantly by its binding to the cell membrane via glycosylphosphatidylinositol (GPI); these netrins present variants that are presumably generated by alternative splicing (Nakashiba et al. 2000). Currently, some Netrin family members have isoforms. Two isoforms of Netrin-1 have been identified (Delloye-Bourgeois et al. 2012; Harter et al. 2014; UniProt Consortium 2018). In the literature and protein databases, only one Netrin-3 isoform has been described (Van Raay et al. 1997). Netrin-4 has five isoforms, which are produced by alternative splicing (Delloye-Bourgeois et al. 2012; UniProt Consortium 2018; Zhang et al. 2004). Netrin-5 has two human Netrin-5 isoforms and one potential human Netrin-5 isoform that is computationally mapped (UniProtKBQ8 WTR8) (UniProt Consortium 2018; Gerhard et al. 2004). Netrin-G1 has at least nine different mRNA isoforms resulting from its complex alternative splicing and a truncated alternative splice isoform (Orozco and Edbauer 2013; Meerabux et al. 2005). Netrin-G2 has been studied very little, and only one isoform was detected (Meerabux et al. 2005; Amira et al. 2004). Members of the Netrins family are structurally similar to laminin and are related to their β or γ short arms. All members of the Netrins family except Netrin-5 include amino-terminal domains V and VI, which are relevant to laminin, which has the corresponding amino-terminal domains. Domain V consists of cysteine-rich epidermal growth factor-like repeats. The domain C in netrins has many positively charged basic residues, while Netrin-Gs (Nakashiba et al. 2002) has a glycosylphosphatidylinositol (GPI) anchor at its C-terminus; it binds to the cell membrane by combining GPI and then performs its biological function. Netrin-4 derives its English name from Sanskrit, named after the Sanskrit word "netr", which means "guide". It is the fourth member of the Netrins family of vertebrates. In 2000, in the process of studying laminin expressed in the nervous system, Yin et al. discovered a sequence of prominent homology to the amino terminal region of the laminin β chain. While amplification of this sequence, they found isolated cDNAs encoding 628 amino acids. It was found to be similar in secondary structure and size to the known netrins, so this new gene was named Netrin-4 (Yin et al. 2000). In the same year, Koch et al. found that (Koch et al. 2000), different from other netrins, this novel netrin was more closely related to the laminin β-chain, so named β-netrin. It was found to be highly expressed in the heart, kidney, retina, astrocytes, ovary and olfactory bulb and proved to be a component of the basement membrane, existing on the basement membrane of blood vessels, ovary and kidney. Later studies found that it is also expressed in neural stem cells (NSCs) (Staquicini et al. 2009). Moreover, β-netrin is expressed in the vomeronasal nerve and lateral olfactory tract of the brain and can promote the growth of neurites in cultured olfactory bulb explants (Koch et al. 2000). The structure of Netrin-4 starts from the amino terminus as a signal peptide, followed by domains similar to laminin globular domain VI, named the laminin N-terminal domain (LamNT), three EGF-like repeat domains, named EGFL, and finally, the C-terminal domain rich in basic amino acids, named the netrin C-terminal domain (NCT) (Fig. 1) (Cirulli and Yebra 2007). Although the crystal structure of Netrin-4 has been clarified (Reuten et al. 2016; Sandoval et al. 2020), studies on Netrin-4 are still mainly focused on its expression distribution, the guidance of axons, angiogenesis, and the occurrence and development of tumors (Lambert et al. 2012; Yuan et al. 2011; Zhang et al. 2004). Until now, the role of Netrin-4 has been unclear. The function of Netrin-4 is closely relevant to the binding of receptors, and its binding site with some receptors has been clarified in crystal structure (Reuten et al. 2016; Sandoval et al. 2020). There are two main types of receptors in the Netrins family: one is the Deleted in Colon Cancer (DCC) family (Keino-Masu et al. 1996) and Neogenin (Kolodziej et al. 1996), the other is Uncoordinated locomotion 5 (Unc5) (Keleman and Dickson 2001) and the newly discovered receptor Down’s syndrome cell adhesion molecule (DSCAM) (Ly et al. 2008), Adenosine receptor A2B (A2BAR) (Corset et al. 2000) and Integrin (Li et al. 2012). Both DCC and Neogenin are type I transmembrane receptors that belong to the immunoglobulin (Ig) superfamily of proteins (Keino-Masu et al. 1996). They have a similar region architecture containing four Ig domains at the N-terminus followed by six fibronectin type III (FNIII) domains, a single-pass transmembrane region and a large cytoplasmic tail that contains three highly conserved sequence motifs named P1, P2 and P3 (Lai Wing Sun et al. 2011). The Unc5 homolog family is a single-spanning transmembrane protein that is composed of four members, Unc5A, Unc5B, Unc5C and Unc5D, in humans (also called Unc5H1, Unc5H2, Unc5H3 and Unc5H4 in rodents). Its extracellular region consists of two Netrin-1 binding Ig domains and two type I thrombospondin domains (TSP) (Geisbrecht et al. 2003). A ZU-5 domain, a DCC-binding (DB) motif and a death domain (DD) motif comprise the cytoplasmic region (Hofmann and Tschopp 1995). DSCAM is a type I TM orphan receptor comprising ten Ig and six fibronectin type III (FNIII) repeats in its extracellular domain and an intracellular domain with no identifiable motifs (Yamakawa et al. 1998). A2BAR was first identified and cloned in 1992 by Rivkees and Reppert and by Pierce et al. from the rat hypothalamus (Rivkees and Reppert 1992) and human hippocampus (Pierce et al. 1992). The proposed structure of A2BAR is the typical G-protein-coupled receptor (GPCR) structure, and the predicted molecular mass of A2BAR is 36–37 kDa (Feoktistov and Biaggioni 1997). Integrin heterodimers are composed of noncovalently associated α and β subunits (Hynes 2002). In vertebrates, the family is composed of 18 α subunits and 8 β subunits that can assemble into 24 different heterodimers (Takada et al. 2007). Of these receptors, DCC, Neogenin, Unc5 and Integrin are all dependent receptors (Thibert and Fombonne 2010; Stupack 2005). Indeed, dependence receptors trigger positive signaling on survival, differentiation and migration in the presence of their ligands, while they transduce a negative signal of cell death induction in the absence of their ligands (Mehlen and Goldschneider 2005). Thus, cells expressing these receptors are in a state of dependence on their respective ligands, which is why these receptors have been named dependence receptors. However, there is still much controversy about the function and mechanism of Netrin-4. In this paper, we first introduced the basic structure of Netrin-4 and its family, then systematically summarized the biological functions of Netrin-4 in axonal guidance, angiogenesis, tumor development and other aspects, and described the possible mechanism of its effects and future research.

Searches were carried out in the electronic databases of Web of Science and NCBI database in March 2022 incorporating search terms such as Netrin-4 and NTN4. Original research articles involving functional studies of Netrin-4 were selected, and biographical-item, meeting abstract, news item, reviews, clinical trials, book chapters, and case reports were excluded. After preliminary database retrieval, 730 related articles were obtained, and 648 articles remained after eliminating duplicate articles. Ninety-seven articles were further screened out according to the inclusion and exclusion criteria. After carefully reading and considering the relevance and reference value of the articles, a total of 74 relevant articles were screened out. Any differences were resolved through discussion and consensus. Function and Possible Mechanism of Netrin-4 Axon Guidance by Netrin-4 Netrins are a secreted protein family whose structures are similar to laminin. They play a role in cell migration and guide axon growth in the development of the central nervous system. As a newly discovered family member, it is expressed in the brain, spinal cord and peripheral nervous system, such as the dorsal root ganglia (Yin et al. 2000), and it is speculated that it plays a guiding role in the growth of axons. Due to the similarity between Netrin-4 and the Netrins family, the researchers examined the effects of Netrin-4 on axons, focusing on the number and length of axons, and found a dose-dependent increase in Netrin-4 (Koch et al. 2000). However, the regulatory mechanism to promote axon growth is still unclear. Netrin-4’s localization in the basement membrane reveals that it is likely to serve as an element for stable growth and maturation or provide positive growth guiding along established axons. Qin S et al. embedded the cortical explants of neonatal Wistar rats in collagen gels containing Netrin-4 and found that the axons extended for the region only limited to the side with a higher concentration of Netrin-4 (Qin et al. 2007). In corneal stroma extracellular matrix, Netrin-4 may affect nerve growth cone and keratocyte behavior (Conrad et al. 2010). When Netrin-4 was added to the thalamus and cortex culture medium, thalamocortical (TC) axon branching was more elaborate, and the total branch length increased. In contrast, elaborate axon branching was reduced in the Netrin-4 knockout rat cortex. Netrin-4 is involved in normal axon branching in the thalamic cortex in vivo and in vitro. His-tagged Netrin-4 was found to bind strongly to Unc5B-expressing cells by a cell surface binding assay. Unc5B is expressed in thalamic cells and participates in axon branching as a receptor for Netrin-4 through further study using organotypic coculture preparations (Hayano et al. 2014). Gilles de la Tourette syndrome (GTS) is a child hood-onset neurodevelopmental disorder characterized by motor and vocal tics (Paschou et al. 2013). Netrin-4 is an attractive candidate susceptibility gene for GTS because its interactions with encoded proteins of WNT and SLIT genes lead the growth cone toward its target (Padmanabhuni et al. 2016; Michaelson 2017; Georgitsi et al. 2016). Rare variants in the Netrin-4 gene could be a contributing factor to the development of sporadic Meniere’s disease (MD) (Gallego-Martinez et al. 2020). Meniere disease is a chronic disorder of the inner ear characterized by the core phenotype of spontaneous, episodic vertigo, associated with low-frequency to middle-frequency sensorineural hearing loss (SNHL), tinnitus and aural fullness (Lopez-Escamez et al. 2015). The Netrin-4 gene consists of 10 exons in its canonical form, and most of the damaging variants are clustered in the second exon of the main canonical form. Netrin-4’s C-terminal signal domain could produce relevant biological effects in the protein. However, the mechanism of axon guiding by Netrin-4 is largely unknown. It has been proposed to act as guidance cues not only on its own but also disturbs basement membranes and laminin networks through high-affinity binding to the laminin γ1 chain (Reuten et al. 2016); nevertheless, in fact, how physiological levels of Netrin-4 actually modulate basement membranes and how in pathological conditions, up- or down-regulation of Netrin-4 directly affects the BMs and thus the cell behavior in this Netrin-4 high/low environment. It remains to be investigated further to confirm this statement. In addition, Netrin-4’s widespread expression in adult rat brains shows that Netrin-4 not only plays a great role in guiding axons during embryonic development but also plays a critical role in neuroplasticity in the adult stage (Zhang et al. 2004). Netrin-4 in a Physiological Context In a physiological context, Netrin-4 participates in the regulation of various physiological processes, such as lung development and salivary gland branching. It is expressed at the proximal end of the mouse lung airway in the basement membrane but not at the distal end. It may prevent ectopic budding and fine-tuning the size and shape of buds emerging by inhibiting ERK kinase through Unc5B (Liu et al. 2004). Netrin-4 is a basement membrane-associated protein (Koch et al. 2000), and it is therefore possible that Netrin-4 and laminins regulate each other’s activities. The expression of Netrin-4 in the sublingual part of the salivary gland was weak in newborn mice. It retards salivary gland branching morphogenesis. Netrin-4 expression took on a network-like pattern of increasing age that integrates into the laminin polymer through interactions with the short arms of lamininγ1 and γ3. The binding is mediated through the laminin N-terminal domain of Netrin-4 (Schneiders et al. 2007). Netrin-4 has an inhibitory function on branching morphogenesis and is increasingly expressed in postnatal development, suggesting a role for Netrin-4 in late stages of embryonic development, in maturation, and in tissue homeostasis. Netrin-4 may also play a role in regulating tissue regeneration and contribute to tissue stability by preventing hyperplasia. In alveolar bone osteoblasts, GO analysis and Western blot confirmed the expression of Netrin-4 (Dangaria et al. 2011), but the effect of Netrin-4 on these proteins has not been further studied. Netrin-4 and Angiogenesis Angiogenesis refers to the formation of new blood vessels from the development of existing capillaries or postcapillary veins. It mainly includes vascular basement membrane degradation during activation; activation, proliferation, and migration of vascular endothelial cells; and reconstruction to form new blood vessels and the vascular network. It is a complex process involving multiple molecules. The factors involved in angiogenesis mainly include matrix metalloproteinase (MMP), vascular endothelial growth factor (VEGF), and endostatin (ENS) (Folkman 1995). Under normal circumstances, angiogenesis is the homeostatic regulation between proangiogenic factors and inhibitors. Once the balance is disturbed, the vascular system is activated, causing excessive angiogenesis or inhibiting the vascular system and leading to vascular degeneration. Netrin-4 Promotes Angiogenesis Netrin-4 is involved in angiogenesis and the formation of vascular networks (Kim et al. 2019), and it acts as a regulator in angiogenesis. In fact, both promoting angiogenesis and antiangiogenic effects of Netrin-4 have been reported. As early as 2009, Hoang S et al. found that intracerebroventricular injection of Netrin-4 increased endothelial proliferation and blood vessel density and improved behavioral recovery from 1 week after cerebral ischemia but had no effect on infarct size or blood–brain barrier permeability. The expression of DCC was significantly increased in peri-infarct cortical neurons from days 1 to 2 after cerebral ischemia. These discoveries suggest that DCC may play an effect on promoting angiogenesis by Netrin-4 and improving poststroke functional recovery by enhancing blood vessel proliferation (Hoang et al. 2009). Meta-analysis showed that Netrin-4 is associated with the prognosis of ischemic stroke (Soderholm et al. 2019); it could thus play a role in the plasticity of axonal growth and synaptic structure recovery after stroke. NSCs are also a promising option for patients with stroke/cerebral ischemia due to their self-regeneration, differentiation and multifunctional properties (Trounson and McDonald 2015). Stem cells reside in specialized microenvironments or “niches” that regulate their self-renewal and differentiation activities. Neurogenesis occurs in discrete areas of the brain, the subventricular zone (SVZ) and the hippocampus, where neurogenic niches have been identified and characterized (Bayer et al. 1982; Taupin 2006). After cerebral ischemia, activation of endogenous NSCs in the SVZ and subgranular zone (SGZ) could produce neurotrophic factors that reduce inflammatory responses and simultaneously promote angiogenesis by activating proangiogenic complexes such as Netrin-4, thus reducing brain injury elicited by hypoxia/ischemia (Staquicini et al. 2009; Lee et al. 2018). Netrin-4 could bind to the laminin γ1 chain, and this molecular complex further interacts with α6β1 integrin on the surface of NSCs. This finding suggests that Netrin-4 is a regulatory factor in NSC biology and that the multimeric complex functions as an integrated supramolecular switch that regulates NSC fate (Staquicini et al. 2009). Netrin-4 promoted the proliferation, migration, and differentiation of endothelial progenitor cells (EPCs) and enhanced neovascularization in a mouse hindlimb ischemia model. Unc5B was expressed in EPCs, and silencing Unc5B reduced the chemotaxis and proliferation of vascular endothelial cells and eliminated the ability of Netrin-4 to promote neovascularization, suggesting that Netrin-4 promotes neovascularity through the activation of Unc5B in EPCs (Lee et al. 2020). In 2006, a paper published in Science detailed the effects of netrins on angiogenesis. They discovered that Netrin-1, -2, and -4 all stimulated the migration of human microvascular endothelial cells (HMVECs) in a dose-dependent fashion and stimulated endothelial proliferation and tube formation. Subsequently, they compared the ability of netrins and VEGF to promote angiogenesis and reperfusion with hindlimb ischemia in a mouse model. Laser Doppler imaging showed that hindlimb perfusion was significantly improved 7 days postsurgery in mice injected with Netrin-4, Netrin-1, or VEGF. Histological analysis showed a significant increase in capillary density and decreased fibrosis in Netrin-4-, Netrin-1- and VEGF-treated animals after day 28. Nevertheless, fewer vessels were stained in Netrin-4- or VEGF-treated mice than in Netrin-1-treated mice, and the effect of Netrin-4 in promoting angiogenesis was certain. Moreover, with Netrin-4 treatment, nerve conduction velocity improved in db/db mice with microvascular disease and polyneuropathy, suggesting that it has potent effects on both endothelial and neural biology (Wilson et al. 2006). In recent years, scientists have discovered that Netrin-4 and its receptors exist in vascular smooth muscle cells and endothelial cells, and Netrin-4 has a role in guiding ECs in angiogenesis. Netrin-4 stimulated bovine retinal endothelial cells (BRECs) to increase the phosphorylation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK)1/2 and p38 in a dose-dependent manner. In BRECs, two receptors for netrins were detected. They are Unc5H1 and DCC, and activation of ERK1/2 induced by Netrin-4 is at least partially required for DCC (Lange et al. 2012). The MAP kinases ERK-1 and ERK-2 play a critical role in regulating the proliferation of retinal ECs (Bullard et al. 2003), and there is evidence that these signaling molecules contribute at least in part to their migration (Huang et al. 2004). It is well known that angiogenesis is mediated through the VEGF receptor, leading to proliferation, vascular permeability, cell migration and cell survival (Ivy et al. 2009). These biological effects involve activation of multiple signaling pathways, such as the Ras/MEK/ERK pathway, leading to cell proliferation; the focal adhesion kinase pathway, leading to focal adhesion turnover and cell migration; activation of the PI3K/Akt pathway, maintaining cell survival and leading to vascular cell permeability; and activation of the MKK4/JNK1/2 pathway, leading to cell survival (Ivy et al. 2009; Salameh et al. 2005). It has been confirmed that FAK, Akt, JNK1/2 and ERK1/2 are phosphorylated and activated after Netrin-4 stimulation in zebrafish vessel formation, providing evidence that these pathways are involved in Netrin-4-mediated angiogenesis in vitro and in vivo (Lambert et al. 2012). Netrin-4 Inhibits Angiogenesis Although studies have confirmed that Netrin-4 has a proangiogenic effect (Padua et al. 2018), more studies have found that Netrin-4 is a negative regulating factor of angiogenesis. In vascular endothelial cells (ECs), Netrin-4 is highly expressed and inhibits VEGF-mediated ECs migration and blood vessel formation (Finney and Orr 2018). To explore the potential effect of Netrin-4 on angiogenesis, Lejmi E provided evidence that Netrin-4 has antiangiogenic activity in three different in vivo models: the Matrigel plug assay, the choroidal neovascularization mouse model, and the tumor angiogenesis model. They found that Netrin-4 was specifically overexpressed in VEGF-stimulated ECs in vitro and in vivo. Knockdown of Netrin-4 expression in ECs increased their ability to form tubular structures on Matrigel. In addition, ECs expressed Neogenin, Unc5B, and Unc5C. Silencing of Neogenin or Unc5B abolished the inhibitory effect of Netrin-4 on ECs migration, suggesting that both receptors are essential for Netrin-4’s function in vitro. They transfected a prostatic cancer cell line with an expression vector encoding Netrin-4. They found that overexpression of Netrin-4 could reduce vascular density and is an inhibitor of tumor angiogenesis, while Netrin-4 secreted by cancer cells was found to reduce angiogenesis by acting on other cells, such as perivascular cells (Lejmi et al. 2008; Fujita and Yamashita 2017). Further studies by this team proved that the structure and grade of blood vessels in tumors transfected with Netrin-4 are closer to the microvascular network of healthy tissues compared with the control group. This finding indicates that Netrin-4 enhances the recruitment of perivascular cells to tumor ECs and induces the maturation of tumor blood vessels, which is essential for vascular normalization (Lejmi et al. 2014). Eveno C et al. confirmed that Netrin-4 was expressed in human colon cancer cells (LS174). A Matrigel angiogenesis assay demonstrated that Netrin-4 overexpression altered VEGF/basic fibroblast growth factor induction angiogenesis. When Netrin-4 LS174 human colon cancer cells were s.c. injected into nude mice, the tumor volume was significantly smaller. Moreover, this effect may be achieved by binding to its receptor neogenin, suggesting that Netrin-4 has potential therapeutic value in the treatment of carcinogenesis and colorectal cancer growth (Eveno et al. 2011, 2013). Netrin-4, although, has shown antitumor potential, but its mechanism is still not clear. An increasing number of studies have confirmed that Netrin-4 also plays a great role in retinal angiogenesis and embryonic development (Alizadeh et al. 2018; Smith et al. 2018). The level of Netrin-4 in cord blood was significantly increased with increasing gestational age, especially in fetuses with abnormal placental angiogenesis (Boutsikou et al. 2014). However, in human placental microvascular endothelial cells (HPECs), Netrin-4 inhibited HPECs proliferation and migration. The role of Netrin-4 in HPECs sprouting was investigated by a three-dimensional in vitro angiogenesis system. It was confirmed that sprouting significantly decreased in HPECs spheroids incubated with Netrin-4. Moreover, the elevation levels of Netrin-4 are correlated with elevation of sFlt-1, an anti-angiogenic agent (Icen et al. 2020), which brought new insights into the role of Netrin-4 in placental angiogenesis (Dakouane-Giudicelli et al. 2015). Human umbilical vein endothelial cells (HUVECs) express Netrin-4 (Prieto et al. 2017), and Netrin-4 inhibits the tube formation of HUVECs in an in vitro angiogenesis assay (Han et al. 2015). The regulation of angiogenesis by Netrin-4 is, at least in part, related to Notch1 signaling. Notch signaling is highly conserved among species and is critical for proliferation, differentiation and fate determination. Mammals possess four Notch receptors (Notch1-4), of which Notch1 and Notch4 are abundantly expressed in ECs. Notch1 is an essential regulator of embryonic vascular development. Both loss- and gain-of-function studies have demonstrated that deregulation of Notch1 results in severe vascular defects (Gridley 2007). Activation of Notch signaling significantly upregulated Netrin-4 mRNA and protein expression in endothelial-specific NICD1 transgenic (Tg) mice and ECs culture models, and the direct binding of Notch1 occurs at the 53 bp TGGGAA site of the Netrin-4 promoter (Liu et al. 2019). The cornea, the absence of blood vessels, is required for optical clarity and optimal vision and has led to the cornea being widely used for validating pro- and antiangiogenic therapeutic strategies for many disorders (Gimbrone et al. 1974; Muthukkaruppan and Auerbach 1979). In contrast to the conclusion that Netrin-4 promoted retinal angiogenesis confirmed by previous studies (Lange et al. 2012), Netrin-4 treatment led to only a small amount of blood vessels in the limbus but not in the peripheral or central corneas in corneal neovascularization induced by alkali burns (Han et al. 2015). NF-ĸB is a family of heterodimeric proteins involved in the regulation of immune and inflammatory responses, apoptosis, and oncogenesis (Karin et al. 2002). Several stimuli, such as cytokines and bacterial products, activate NF-ĸB, primarily through inhibitor of nuclear factor-B (IB) kinase (IKK)-dependent phosphorylation and subsequent degradation of specific inhibitors called IB proteins (Ghosh and Karin 2002). Once activated, NF-ĸB dimers enter the nucleus and modulate the transcription of many genes encoding cytokines, cell adhesion molecules, and antiapoptotic proteins. Pigment epithelium-derived factor (PEDF), a potent anti-angiogenic factor, is found in retinoblastoma cells, retinal pigment epithelia, iris and cornea (Ortego et al. 1996). It can promote ECs apoptosis and inhibit ECs migration and tube formation (Becerra 2006). It is well established that corneal neovascularization is tightly regulated by a dynamic, natural equilibrium between local proangiogenic and antiangiogenic molecules (Amano et al. 1998; Ambati et al. 2006; Mwaikambo et al. 2006; Stuart et al. 2003). Among them, VEGF and PEDF are considered to play major roles. Netrin-4 inhibited VEGF and the cell death of corneal epithelial and stromal cells but promoted the expression of PEDF, reduced NF-ĸB p65 expression, and inhibited the infiltration of neutrophils and macrophages (Han et al. 2015). This demonstrated that Netrin-4 can restore the disrupted balance between VEGF and PEDF as an antiangiogenic effect. These results reveal that Netrin-4 has a novel effect in the treatment of angiogenesis in the cornea and other tissues, which also indicates that its expression in the corneal basement membrane is of some significance (Kabosova et al. 2007). In Ntn4-deficient mice (Ntn-4−/−), as the gestational age of mice increases, the avascular area of the retina decreases compared with WT mice. Netrin-4 is necessary for maintaining the stabilization of mature blood vessels, and it needs to balance with VEGF expression to play a role in physiological and pathological angiogenesis (Han et al. 2015; Kociok et al. 2016). Although the receptor of Netrin-4 is not yet clear, studies have shown that Unc5B (= Unc5H2), Unc5C, Unc5D, Neogenin, DCC, and A2B are expressed in the retinas of mice (Liu et al. 2014). However, only Unc5H2 was upregulated after oxygen-induced retinopathy (OIR). DCC immunoreactivity appeared to colocalize with GFAP, suggesting that astrocytes or Müller cells in the inner retina could respond to changes in the basal membrane with Netrin-4 expression (Kociok et al. 2016). In a mouse model of suture-induced inflammatory corneal neovascularization, Netrin-4 was strongly expressed in the corneal epithelium, endothelial basement membrane, and corneal vascular basement membrane (Maier et al. 2017). Knocking out Ntn4 significantly increased the vascular pedicle area of Ntn4−/− mice compared with WT mice. When corneal epithelial cells or corneal keratocytes were stimulated with different concentrations of VEGF-A (VEGF family members) or Netrin-4, the expression of VEGF-A, Ntn4, Unc5H2 or Neogenin mRNA was not influenced. These results suggest that these cells did not show autocrine regulation of Netrin-4 expression by either Netrin-4 or VEGF-A. However, Ntn4 deficiency resulted in an augmentation of VEGF-A mRNA expression driven by angiogenesis. Therefore, it can be assumed that corneal epithelial cells indeed enrich the basement membrane by expressing Netrin-4 and establish an antiangiogenic environment in the cornea(Maier et al. 2017). In agreement with previous studies (Han et al. 2015; Kociok et al. 2016), the antiangiogenic effect of Netrin-4 is closely correlated with the expression of VEGF (Maier et al. 2017). On the other hand, researchers seeded human dermal microvascular endothelial cells (HDMECs) into Matrigel and added different concentrations of Netrin-4 after cell attachment to Matrigel. Then, Netrin-4 completely eliminated tube-like structure formation with the treatment of time and concentration of Netrin-4. The effect of Netrin-4 destabilized capillaries in vivo, with a significant reduction in the observed vascularized area, and the capillary network changed dramatically. Netrin-4 plays an important role in the regulation of angiogenesis in pathological retinas. Studies have shown that a lack of Ntn4 leads to an increased tortuosity of blood vessels and that inner retina function appears to be affected. In addition, Ntn4 deficiency resulted in spontaneous leakage and a higher persistence of hyaloid arteries in the adult retina. Remarkably, no differences were found in vessel width, vessel bifurcation, or vein/artery ratio compared with WT mice (Crespo-Garcia et al. 2019). The same group later identified that Netrin-4 provided a protective environment for inflammation in the diabetic retina, blocking the production of cytokines, and that Ntn4 deficiency exacerbated inflammation in diabetic retinopathy (Crespo-Garcia et al. 2021). This is in agreement with previous findings that increased Netrin-4 alleviates vascular lesions in diabetic retinopathy (Cao et al. 2017). These studies reveal that Netrin-4 plays a crucial role in maintaining vascular homeostasis. On the basis of the above studies, some have defined Netrin-4 as an angiogenic promoter, while others have suggested the opposite. Further studies are needed to uncover the mechanism by which Netrin-4 affects vascular remodeling. Netrin-4 and Tumors An increasing number of studies have shown that Netrin-4 is also involved in the development of tumors (Hao et al. 2020) and modulates tumor cell proliferation (Hu et al. 2012), metastasis (Larrieu-Lahargue et al. 2010), and invasion (Jayachandran et al. 2016). In particular, Netrin-4 is an independent predictor of outcome in some tumors (Esseghir et al. 2007), and the forms of regulation of tumors are also different. Netrin-4 and Breast Cancer Netrin-4 is expressed in the normal breast and all breast tumors (Esseghir et al. 2007). In normal breast tissue, Netrin-4 was mainly localized to the basal lamina around the epithelial structures and blood vessels. Immunostaining of a series of breast tumors was consistent with the in situ hybridization results of Netrin-4 in normal breast tissues. In addition, 37.5% of invasive breast tumors were positive for Ntn4 transcription, and these results suggest that the expression of Netrin-4 in breast cancer may be a potential prognostic marker. Its overexpression in mouse and human breast cancer cells leads to enhanced metastasis. After subcutaneous xenotransplantation of MCF-7 cells in low invasive human breast cancer or in situ transplantation of 66c14 cells in metastatic mouse breast cancer, Netrin-4 was detected only in MCF-7 cells that were stably transfected with vector encoding mouse Ntn4. In vivo, Netrin-4 induced the growth of blood and lymphatic vessels in the skin and mammary tumors of transgenic mice. In vitro and in vivo, it stimulates the phosphorylation and lymphatic permeability of the intracellular signaling components Akt, ERK, and S6 by activating small GTPases and Src family kinases/FAK. Therefore, it is suggested that Netrin-4 is a lymphangiogenic factor promoting tumor dissemination and a potential target for inhibiting metastasis formation (Larrieu-Lahargue et al. 2010). The Ntn4 is overexpressed in breast carcinoma effusions compared to primary carcinomas using gene expression arrays, but there is no association between Netrin-4 expression and clinicopathologic parameters or with disease-free or overall survival (Yuan et al. 2011). Nevertheless, Oncomine data showed that Ntn4 was reduced in breast cancer compared to normal breast tissue. Overexpression of Ntn4 in MDA-MB-231 cells attenuated cell invasion and migration and induced downregulation of N-cadherin and Vimentin, while transfection of Hs578T cells with Ntn4 siRNA significantly increased migration and invasion and upregulated the expression of N-cadherin and vimentin. These studies suggest that Netrin-4 is involved in breast cancer cell migration and invasion by regulating epithelial mesenchymal transformation (EMT)-related biomarkers (Xu et al. 2017). In addition, Netrin-4 and α6β1 costaining was detected in ECs in MCF7 cells, indicating that a noncanonical netrin receptor, α6β1 integrin, played a role in Netrin-4 function in regulating angiogenesis, tumor growth and dissemination (Larrieu-Lahargue et al. 2011). Inhibition of α6 or β1 integrin subunits suppresses Netrin-4-induced endothelial cell migration, adhesion and focal adhesion contact. α6β1 integrin is a Netrin-4 receptor in the lymphatic endothelium and consequently represents a potential target to inhibit Netrin-4-induced metastatic dissemination. The expression of Netrin-4 was elevated in Unc5A knockdown breast cancer cells (MCF7 and T-47D cell lines), and lower expression of Unc5A was associated with poor outcome (Padua et al. 2018). Combining the above research, not only Netrin-4 but also the receptor of Netrin-4 is closely related to breast cancer. However, there are no studies investigating the relationship between other receptors and breast cancer. In recent years, with the increasing attention of researchers on microRNAs (Simonson and Das 2015), Netrin-4 mRNA was the possible target site of miR-17-5p. miR-17-5p was upregulated in breast cancer specimens in The Cancer Genome Atlas (TCGA) compared with normal breast samples. Netrin-4 mRNA and protein were downregulated after overexpression of miR-17-5p in Hs578T cells, whereas inhibition of miR-17-5p had the opposite effect on MCF-7 cells. This finding indicates that miR-17-5p reduces Netrin-4 expression. TargetScan and miRDB, miRNA databases, predicted that a sequence in the 3′-UTR of Netrin-4 mRNA was a binding site of miR-17-5p. Research data also confirmed this prediction, confirming that miR-17-5p directly suppressed Netrin-4 expression in human breast cancer by targeting the 3′-UTR of Netrin-4 mRNA (Wang et al. 2019). Most studies have reported on the tumor-promoting and antitumor activities of Netrin-4; however, all of these studies focused only on the direct effects of Netrin-4 on cells (Villanueva et al. 2017; Xu et al. 2017). Netrin-4 is a key regulator of basement membrane stiffness. A high level of Netrin-4 is significantly associated with a good prognosis of breast cancer. The ratio of Netrin-4 to laminin determines the stiffness of the basement membrane. The more Netrin-4 there is, the softer the basement membrane, thereby reducing the invasive activity of cancer cells (Mehlen and Fattet 2021; Reuten et al. 2021). Netrin-4 is closely related to the metastasis and invasion of breast cancer (Larrieu-Lahargue et al. 2011; Wang et al. 2019; Gorbatenko et al. 2019). Data from genome-wide association studies (GWASs) showed that suppressed Netrin-4 increases cancer-related processes, including cell proliferation and tumor growth (Beesley et al. 2020). These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition. However, a survival analysis of breast cancer revealed that high expression of Netrin-4 was correlated with poor prognosis of human epidermal growth factor receptor type 2 (HER2)-positive breast cancer patients (Kuroiwa et al. 2020). HER2-positive breast cancer is a subtype of breast cancer (Fragomeni et al. 2018). HER2 overexpression is observed in approximately 25–30% of breast cancers (Slamon et al. 1989; Cobleigh et al. 1999). We acknowledge that further functional studies will be required to clarify how Netrin-4 contributes to breast tumor development. Netrin-4 and Glioblastoma Glioblastoma is the most common malignant tumor in the central nervous system (Louis 2006). Assessment of gene expression profiles of human glioma cells derived from tumor core and white matter invading cell populations from paired patients by laser capture of glioblastoma multiforme (GBM) cells with Hoelzinger, DB et al. It confirmed that Netrin-4 expression is upregulated in invasive glioblastoma (Hoelzinger et al. 2005), it was detected in three invasive glioblastoma cell lines: U373MG, U251MG and U87MG, and suppression of Netrin-4 could reduce cell proliferation and motility, and increase cell death caused by serum deprivation. Integrin β4, the β-subunit of integrin heterodimers, is predominantly expressed in epithelial cells. It is upregulated during tumor progression and pathologic angiogenesis (Giancotti 2007). In the CNS, integrin β4 is expressed strongly in astrocytes, and it is upregulated during glioma progression (Previtali et al. 1996; Shaw et al. 1996). Using tandem affinity purification combined with mass spectrometry protein identification, the study revealed that integrin β4 interacted with Netrin-4. U251MG cells displayed concentration-dependent activation of Akt and mTOR after exogenous addition of Netrin-4. This suggested that Netrin-4 promotes glioblastoma cell growth by stimulating the phosphorylation of the Akt-mTOR signaling pathway. Strikingly, Netrin-4 was not able to activate Akt in integrin β4-suppressed U251MG cells, indicating that integrin β4 mediates Netrin-4-induced Akt-mTOR phosphorylation. Importantly, analysis of more than 400 primary tumors from The Cancer Genome Atlas repository revealed that Netrin-4 is significantly downregulated and integrin β4 is upregulated in glioblastoma, which is associated with poor patient survival time (Hu et al. 2012). Some studies also support the above conclusions, discovering that Netrin-4 can rescue the decrease in the phosphorylation of Akt, ERK and mTOR caused by temozolomide (TMZ). Temozolomide, the 3-methyl derivative of mitozolomide, has comparable antitumor activity in preclinical studies, a favorable toxicological profile, and excellent oral bioavailability and tissue distribution, including central nervous system penetration (Newlands et al. 1992; Stevens et al. 1987). Under physiological conditions, temozolomide is rapidly hydrolyzed and converted into 5-(3-methyltriazeno)imidazole-4-carboxamide (MTIC), the active agent (Tsang et al. 1991). MTIC is subsequently hydrolyzed to 5-amino-imidazole-4-carboxamide (AIC), which, when bound as a ribonucleotide, is an intermediate in the generation of inosine monophosphate and hence an intermediate compound of purine biosynthesis. AIC is additionally converted to a methyldiazonium cation that is capable of alkylating DNA, again at the O6 and N7 positions on guanine and the N3 position on adenine (Villano et al. 2009; Newlands et al. 1997). Alkylation at the O6 position of guanine leads to mispairing with thymine, and continual mispairing leads to double-stranded breaks and apoptosis of the affected cell. Overexpressing integrin β4 in combination with exogenous recombinant Netrin-4 could prevent TMZ-induced cellular senescence in U87MG cells, indicating that interfering with the interaction between integrin β4 and Netrin-4 or concomitantly using Akt pathway inhibitors may improve the therapeutic efficiency of TMZ. This result revealed that integrin β4 mediates the protective effect of Netrin-4 on TMZ-treated glioblastoma cells (Li et al. 2013). Subsequent research by this team found that EGFR and its related genes are involved in extracellular matrix (ECM) organization, cell adhesion and caspase-related signaling. It stimulated the expression of Netrin-4 in GBM cells and cooperated with Netrin-4 to attenuate DNA damage-induced senescence in GBM cells, possibly through Akt and ERK (Li et al. 2018a). This also provides important evidence for subsequent studies on the anti-senescence effects of Netrin-4 (Zhang et al. 2021). Netrin-4 and Colorectal Cancer Colorectal cancer is a common malignant tumor in the gastrointestinal tract. According to the statistics of the world cancer epidemiological survey, its incidence is high in North America and Western Europe (Lieberman 2010), which may be related to the dietary habits of residents. Netrin-4 is expressed in LS174 human colon cancer cells, inhibits colorectal tumor growth and impairs tumor angiogenesis. It has no direct effect on the proliferation and cell death of tumor cells; instead, it regulates tumor cell proliferation and cell death by mediating the antiangiogenic activity of Netrin-4 through its receptor Neogenin (Eveno et al. 2011). The research team later demonstrated in the orthotopic liver metastasis model that Netrin-4 overexpression reduces colorectal metastasis and its associated lymph node involvement. It decreases tumor metastasis and recurrence after surgical resection, possibly via an antiangiogenic effect (Eveno et al. 2013). Therefore, Netrin-4 may have a greater potential for the treatment of colorectal cancer in the future (Xu et al. 2013). Netrin-4 and Other Tumors In addition to the above tumors, there are few studies on Netrin-4 in other tumors, but it also plays an important role. Netrin-4 and its receptor Neogenin are expressed in neuroblastoma, and Neogenin regulates cell death and cell migration through Netrin-4, which is involved in the metastasis of tumor cells in vivo (Villanueva et al. 2017). Forming a ternary complex of laminin γ1 (LMγ1) and Neogenin, Netrin-4 acts as a crucial extracellular matrix component that induces neuroblastoma cell migration (Villanueva et al. 2019, 2021). In metastatic melanoma tumors, Netrin-4 was highly expressed, and silencing Ntn4 led to reduced melanoma invasion in vitro. Moreover, the migration of melanoma cells was suppressed after transplantation of melanoma cells that were transfected with siRNA targeting Ntn4 into the trunk neural tube of a developing chick embryo, which suggests that Netrin-4 plays an important role in promoting melanoma cell invasion (Jayachandran et al. 2016). Nevertheless, in a proliferation assay, the human melanoma cell line SK-MEL-28 is less responsive than many other human tumor lines (Pancreas, Liver, Cervical, Breast and Kidney cell lines) to Netrin-4 protein (Nacht et al. 2009). In melanoma, invasion and proliferation are uncoupled, such that highly proliferative melanoma cells are less likely to be invasive, and vice versa (Jayachandran et al. 2014; Hoek et al. 2008; Wehbe et al. 2012), but the properties are not mutually exclusive, and invading cells undergo proliferation (Haass et al. 2014). The lncRNA olfactory receptor, family 3, subfamily A, member 4 (OR3A4) is strongly expressed in gastric cancer (GC) tissues. Netrin-4 was identified as a direct target of OR3A4 by global microarray analysis and confirmed by qRT‒PCR, RNA immunoprecipitation, and RNA pull-down assays. OR3A4 overexpression may promote growth, invasion, metastasis, and tumorigenesis in GC in vitro and in vivo by downregulating Netrin-4 expression (Guo et al. 2016). Ntn4 knockdown significantly inhibited cell motility and proliferation, while overexpression or addition of Netrin-4 reversed these effects. In addition, Neogenin, Netrin-4’s receptor, was also found to be highly expressed in GC cells. It mediated Netrin-4-induced cell invasion and proliferation. Moreover, silencing of Netrin-4 or Neogenin decreased the phosphorylation of ERK, Stat3, p38 and Akt, indicating that multiple oncogenic pathways (PI3K/Akt, ERK/MAPK and Jak/Stat) were involved in the induction of GC cells by Netrin-4. Netrin-4 expression was negatively correlated with the survival period and positively associated with the severity of pathological stages of GC. Taken together, these results suggest that Netrin-4 may serve as a potential noninvasive biomarker for the diagnosis and prognosis of GC (Lv et al. 2015). In hepatocellular carcinoma, after treatment with a novel antitumor monoclonal antibody (AC10364) in hepatocellular carcinoma cells, Netrin-4 expression was downregulated, suggesting that Netrin-4 may be a promising therapeutic candidate for cancer therapy (Wu et al. 2019). Of course, gene expression analysis showed that the level of Netrin-4 was lower in hepatocellular carcinoma tissues than in adjacent nontumor tissues. However, survival analysis suggests that Netrin-4 is not associated with the prognosis of hepatocellular carcinoma (Zheng et al. 2019). In cervical cancer (Zhang et al. 2013), it was detected that the expression of Netrin-4 was also downregulated, and it played a role in regulating cell proliferation and invasion (Lu et al. 2016). Collectively, Netrin-4 may serve as a potential antitumor therapeutic target, which has positive significance for the future treatment of cancer. Netrin-4 in Other Diseases Netrin-4 plays a key role in the normal physiological context, axon guidance, angiogenesis, and tumor development, as well as in the regulation of other diseases. In repeated implantation failure of the endometrium and endometriosis, the expression of Netrin-4 was downregulated (Revel et al. 2011; Zhao et al. 2014; Agrawal et al. 2018), and it is believed that it could serve as a new candidate for the diagnosis and therapy of embryo implantation failures. In glomerular disease, it is often characterized by altered histological patterns of the extracellular matrix. Researchers have examined the expression of proteins in the extracellular matrix of mouse glomeruli and found that the expression of Netrin-4 is upregulated (Randles et al. 2015). It was demonstrated that changes from extracellular matrix components included Netrin-4 (Lennon and Hosawi 2016). Intriguingly, Netrin-4 increases angiogenic tube formation and endothelial cell migration but does not alter proliferation. It plays a crucial synergistic role in the establishment of the glomerular filter (Kim et al. 2019), which is believed to be related to the regulation of glomerular morphology and susceptibility. To quantitatively compare protein abundance between glomeruli of patients with IgA nephropathy (IgAN) and controls with normal biopsy findings, mass spectrometry-based proteomics was used by Paunas, FTI et al. Of the basement membrane proteins, Netrin-4 was significantly more abundant in IgAN compared to controls (Paunas et al. 2019). A logistic recession model was used to calculate the effects of candidate single nucleotide polymorphisms (SNPs) on IgAN risk after adjusting for age and sex differences, which revealed a significant relationship between the homozygotic genotype for Ntn4 rs1362970 A/A and a higher risk of IgAN (Feng et al. 2019). However, further research should be conducted to investigate and validate the mechanism by which the above findings affect IgAN. In 2016, researchers found that Netrin-4 was expressed in dorsal horn inner lamina II excitatory interneurons in the rat spinal cord (Hayano et al. 2016), the majority of which are interneurons that modulate and transmit sensory information from primary afferents (Yasaka et al. 2010). Behavioral analysis revealed that tactile and heat hyperalgesia after peripheral nerve injury or inflammation were abolished in Netrin-4 mutant rats. Transient suppression of Netrin-4 or its receptor Unc5B after injury could also prevent allodynia. Conversely, intrathecal administration of Netrin-4 protein to naive rats enhanced excitatory synaptic transmission in the dorsal horn and induced allodynia, suggesting that Netrin-4 is involved in spinal sensitization. Furthermore, the Unc5B receptor and subsequent activation of the tyrosine phosphatase SHP2 mediated Netrin-4-induced pain signaling in the spinal cord (Hayano et al. 2016). Thus, it is believed that Netrin-4 is a novel protein that regulates spinal sensitization leading to chronic pain. Moreover, Netrin-4 is involved in neuropathic pain of the maxillofacial region. Quantitative mRNA analysis of the trigeminal subnucleus caudalis (Vc)-upper cervical cord (C1) indicated significantly increased Netrin-4 at 14 days after chronic constriction injury (CCI)-Sham injury compared with before injury. This finding indicates that hyperalgesia is caused by increased Netrin-4 secretion, which triggers neuronal activation. Anti-Netrin-4 antibody presents an analgesic effect by inhibiting the binding of Netrin-4 to Unc5B and shows neuronal activity suppression (Honjo et al. 2021). In addition, the expression of axonal outgrowth pathway-related proteins, including Netrin-4, was downregulated in nasal polyps (Wu et al. 2018). RNA sequencing (RNA-seq) and bioinformatics analysis showed antifibrosis long noncoding RNAs (lncRNAs) in activated human HSCs (hHSCs) and found to be associated with Netrin-4 (Li et al. 2018b; Gerhard et al. 2020; Lin et al. 2018), which may serve as a potential target for reversing the progression of liver fibrosis. In addition, the existence of Netrin-4 and its receptor in islet cells was further confirmed by RT‒PCR, Western blot and immunofluorescence staining (Yang et al. 2011). When pancreatic islet cells were exposed to Netrin-4, downregulation of caspase-3 activation was observed. Reduction of caspase-3 cleavages was associated with a decrease in the dependent receptors Unc5A and Neogenin, as well as the activation of Akt and ERK signaling. Interestingly, studies found that Netrin-4 recognition by embryonic pancreatic cells through integrin α2β1 and α3β1 promoted the expression of insulin and glucagon genes. In addition, fetal pancreatic cell adhesion to Netrin-4 caused a significant downregulation of cyclins and upregulation of negative cell cycle regulators (Yebra et al. 2011), and Netrin-4 is an additional ligand for α2β1 and α3β1 integrins that can affect islet cell phenotype. These results suggest that Netrin-4 may be a pro-differentiation cue for neighboring developing pancreatic cell populations that express adhesion receptors α2β1 and α3β1 integrins. In the nearly two decades since Netrin-4 was discovered as a regulator of axonal guidance, it has been studied endlessly in various tissues and organs, and they all play an important role. It is expected to become a target of medical treatment in the future. However, most of the research in other tissues and organs is still at a phenomenon level, and the mechanism of its function still needs to be confirmed by further research.

and Perspectives In conclusion, as one of the highly conserved members of the Netrins family, Netrin-4 plays an important role in more physiological and pathological processes in addition to its role in the central nervous system, including its involvement in the development and metastasis of a variety of cancers, and plays a role in promoting or inhibiting angiogenesis, which makes it an important protein in regulating various biological events. The possible regulatory role of Netrin-4 in angiogenesis and tumorigenesis and development is summarized in Fig. 2 and Fig. 3. There are contradictory findings on the role of Netrin-4 and its receptors in angiogenesis. Inhibition or augmentation of Netrin-4, its receptors or modulation of the interaction between them will provide clinical benefit in different diseases remains to be determined. Targeting Netrin-4 is of great interest because its expression is upregulated in many cancers. Theoretically, it may be combined with other treatment modalities, and the study of the Netrin-4 pathway may expand the list of dependent receptors and tumor suppressors, particularly with today’s advanced molecular studies. In the development of the central nervous system (CNS), in addition to the role of axonal guidance, research on the development of white matter in the brain has not been reported. ECs are the major source of Netrin-4 secretion, and it was recently found that the oligovascular unit (OVU), a structural unit similar to the neurovascular unit (NVU), is present in white matter. It is mainly composed of vascular endothelial cells, neuronal axons, astrocytes and oligodendrocyte precursor cells/oligodendrocytes (OPCs/OLs). Moreover, the oligovascular niche consisting of OPCs, ECs and their secreted cell signals has been shown to be involved in the repair of white matter injury by regulating gliogenesis and angiogenesis (Arai and Lo 2009). In addition, OPCs migrated from the SVZ to other brain regions along the vascular scaffold formed by vascular ECs during CNS development in mice, including embryonic and postnatal migration time. Abnormal development of cerebral blood vessels leads to decreased migration ability of OPCs, and OPCs migration along the blood vessels stops and separates from ECs, resulting in abnormal aggregation into plaques or spheroids (Tsai et al. 2016). Therefore, it is concluded that Netrin-4 secreted by ECs may have certain effects on OLs. It will be interesting to study how Netrin-4 regulates the proliferation, differentiation and migration of OLs. This research gap in these areas requires us to use a variety of methods to conduct further research and reveal them. In addition, in the past two decades, studies on the Netrin-4 receptor have mainly focused on the classical receptors of the Netrins family. As a secreted protein, Netrin-4 is involved in the development and progression of a variety of diseases. In this paper, we summarize the research progress on Netrin-4 and several classical receptors (Table 1). However, the mechanisms underlying the involvement of Netrin-4 and its various receptors in these processes are poorly understood. In addition, whether Netrin-4 has potential specific receptors has not been reported. Future research on the half-life, posttranslational and transcriptional regulation of Netrin-4 and the potential mode of action will be beneficial to our understanding of its effect, which will also be valuable in ultimately providing therapeutic targets for clinical diseases and therapy. Table 1. | References | Samples | Cells | Receptors | Significant outcome | |---|---|---|---|---| | Qin S et al. (Qin et al. 2007) | Newborn Wistar rats | Primary neonatal cortical neurons of Wistar rats | DCC, Unc5H1 | DCC and Unc5H1 are receptors for Netrin-4, only LNT and NCT is the key domain for specific binding | | Lange et al. (Lange et al. 2012) | Guinea Pigs, Rhesus Monkey, Long Evans Rats | Primary guinea-pig and rat Müller cells | DCC, Unc5H1 | Netrin-4 stimulated BRECs to increase phosphorylation of the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK)1/2, and p38, in a dose-dependent manner. DCC and Unc5H1 were present in retinal endothelial cells, and DCC is at least partially required for Netrin-4-induced activation of ERK-1/-2 | | Lejmi et al. (Lejmi et al. 2014) | Nude mice | Vascular smooth muscle cell (VSMC), Human Umbilical Arterial Endothelial Cells (HUAEC), Primary Porcine Retinal Pericyte cells (PRPC) | DCC, Neogenin, Unc5B | Silencing of DCC, Neogenin and Unc5B reduced Netrin-4 adhesion to VSMC, suggesting that these receptors were involved in the recruitment process | | Hoang et al. (Hoang et al. 2009) | C57BL/6 mice | – | DCC | DCC was upregulated in the peri-infarct cortex after stroke, while Netrin-4 was upregulated in astrocytes and vessels in the ischemic core | | Kociok et al. (Kociok et al. 2016) | C57BL/6 mice, Ntn4−/− mice | – | DCC, Unc5H2 | Unc5H2 and DCC were found in Müller cells and astrocytes, Ntn4−/− mice initially displayed larger avascular areas which recovered faster to revascularization | | Lejmi E et al. (Lejmi et al. 2008) | C57BL/6 mice | Human umbilical vein EC (HUVEC), human umbilical artery EC (HUAEC) | Neogenin, Unc5B, Unc5C | silencing of either Neogenin or Unc5B abolished Netrin-4 inhibitory effect on ECs migration, suggesting that both receptors are essential for its function in vitro | | Eveno C, et al. (Eveno et al. 2011) | Athymic nude mice | LS174 human colon cancer cells | Neogenin | Ntn4 overexpression inhibited tumor angiogenesis and growth, the antiangiogenic activity of Netrin-4 was probably mediated by binding to its receptor Neogenin | | Yang YH, et al. (Yang et al. 2011) | C57BL/6 mice | Primary islet cells of mice, MIN6 cells, human islet cells | Neogenin, Unc5A | Netrin-4 downregulates caspase-3 activity, which related to the decrease of Neogenin and Unc5A | | Dakouane-Giudicelli M, et al. (Dakouane-Giudicelli et al. 2015; Dakouane-Giudicelli et al. 2012) | Human placenta | Human placental microvascular endothelial cells (HPEC) | Neogenin | Netrin-4 and Neogenin are highly placenta, and may play an important role in placental angiogenesis | | Lv B, et al. (Lv et al. 2015) | Gastric adenocarcinoma tissue | Human gastric cancer cell lines AGS and MGC803 | Neogenin | Ntn4 or Neogenin silencing decreased the phosphorylation of STAT3, ERK, Akt and p38, indicating that Netrin-4 plays a role through Neogenin | | Villanueva AA, et al. (Villanueva et al. 2017) | – | Neuroblastoma cell lines SH-SY5Y, SKN-SH, LAN-1 andNB1691, U87 (glioblastoma), HEK293 (human embryonic kidney), DAOY (medulloblastoma) | Neogenin | Knocking-down Neogenin reduces cell migration, whereas silencing of endogenous Ntn4 induced cell death | | Villanueva et al. (Villanueva et al. 2019) | Human tumor samples | SK-N-SH cells | Neogenin, Laminin γ1 | Netrin-4, by forming a ternary complex with Laminin γ1 (LMγ1) and NEO1, induces the migration of SK-N-SH cells | | Larrieu-Lahargue et al. (Larrieu-Lahargue et al. 2010) | NOS/SCID mice | Lymphatic dermal Human Microvascular Endothelial Cells (HMVEC-dLys), dermal Human Microvascular Endothelial Cells (HMVEC-ds), Human umbilical vein endothelial cells (HUVECs), Human umbilical artery endothelial cells (HUAECs), Human MCF7 breast cancer cells, Mouse 66C14 mammary carcinoma | Unc5B, Neogenin, Adenosine receptor A2b | Unc5B, Neogenin and A2b are expressed by human lymphatic endothelial cells, suppression of either or both does not suppress Netrin-4 promoted proliferation, migration and phosphorylation of ERK1/2 | | Lee et al. (Lee et al. 2020) | BALB/CA-nu/nu Mice | Endothelial progenitor cells (EPCs) | Unc5B | Unc5B silencing decreased SEPC proliferation and chemotaxis, Netrin-4 stimulation of EPCs is mediated by Unc5B receptor signaling | | Takemoto et al. (Takemoto et al. 2011) | C57BL/6 mice Kidney 293 T (HEK293T) cells | Human embryonic | Unc5D | Netrin-4 protein bound to Unc5D-expressing cells, Unc5D is not sufficient to mediate the effect of Netrin-4 in deep layer cells | | Reuten et al. (Reuten et al. 2016) | C57BL/6 mice | mouse melanoma cell lines HCMel12, B16-F1, HEK293 EBNA | Laminin γ1 | Netrin-4 does not bind directly to any of the known Netrin-1 receptors, Netrin-4/lamininγ1 interaction destabilizes capillaries in vivo | | Yebra et al. (Yebra et al. 2011) | Human pancreatic tissue | Human pancreatic epithelial cells | Integrinα2β1 and α3β1 | Netrin-4 recognition by embryonic pancreatic cells through integrins α2β1 and α3β1 promotes insulin and glucagon gene expression | | Hu et al. (Hu et al. 2012) | – | 293FT cells, Astrocytes, U251MG cells, U87MG cells, U373MG cells | Unc5B, Integrinβ4 | Integrinβ4 interacts with netrin-4, the negative effects of Netrin-4 on proliferation were mediated by Unc5B | | Larrieu-Lahargue et al. (Larrieu-Lahargue et al. 2011) | – | Human endothelial cell (EC) | integrinα6β1 | α6β1 integrin is a Netrin-4 receptor in lymphatic endothelium and consequently represents a potential target to inhibit Netrin-4-induced metastatic dissemination | | Schneiders et al. (Schneiders et al. 2007) | C57BL/6 mice kidney | human embryonic kidney cells (293-EBNA) | lamininγ1, lamininγ3 | Netrin-4 interacts with the Lamininγ1 | | Staquicini et al. (Staquicini et al. 2009) | BALB/cByJ mice | NSCs | Lamininγ1, integrinα6β1 | Netrin-4, Laminin-γ1 and Integrin α6β1 form a ternary complex that mediates astrocyte/neuron interactions |

This work was supported by the National Natural Science Foundation of China [No. 16611744]. Author Contributions FDong and YL : prepared the figures and drafted and wrote the manuscript. WY, QM, XS and BC: proofread and revised the manuscript with the supervision of RY. All authors contributed to the article and approved the submitted version. Funding National Natural Science Foundation of China,16611744 Declarations Conflict of interest All authors declare that they have no conflicts of interest. Footnotes Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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