Neuroinflammatory disease disrupts the blood-CNS barrier via crosstalk between proinflammatory and endothelial-to-mesenchymal-transition signaling.

Breakdown of the blood-central nervous system barrier (BCNSB) is a hallmark of many neuroinflammatory disorders, such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we show that endothelial-to-mesenchymal transition (EndoMT) occurs in the CNS before the onset of clinical symptoms and plays a major role in the breakdown of BCNSB function. EndoMT can be induced by an IL-1ß-stimulated signaling pathway in which activation of the small GTPase ADP ribosylation factor 6 (ARF6) leads to crosstalk with the activin receptor-like kinase (ALK)-SMAD1/5 pathway. Inhibiting the activation of ARF6 both prevents and reverses EndoMT, stabilizes BCNSB function, reduces demyelination, and attenuates symptoms even after the establishment of severe EAE, without immunocompromising the host. Pan-inhibition of ALKs also reduces disease severity in the EAE model. Therefore, multiple components of the IL-1ß-ARF6-ALK-SMAD1/5 pathway could be targeted for the treatment of a variety of neuroinflammatory disorders.

Deletion of Arno Reduces Eosinophilic Inflammation and Interleukin-5 Expression in a Murine Model of Rhinitis.

BACKGROUND: ARF nucleotide-binding site opener (ARNO) is a guanine nucleotide-exchange factor for ADP-ribosylation factor proteins. ARF nucleotide-binding site opener also binds MyD88, and small-molecule inhibition of ARNO reduces inflammation in animal models of inflammatory arthritis and acute inflammation. However, whether genetic deletion of Arno in mice reduces pathologic inflammation has not yet been reported. Furthermore, its role in the nasal cavity has yet to be investigated. OBJECTIVE: To generate Arno knockout mice and to determine whether genetic loss of ARNO reduces eosinophilic inflammation in the ovalbumin (OVA) murine model of rhinitis. METHODS: Arno knockout mice were generated and wild type and knockout littermates were subjected to the OVA-induced mouse model of rhinosinutitis. Eosinophilic inflammation was assessed through immunofluorescent quantification of EMBP+ eosinophils in the septal mucosa and cytokine expression was assessed by quantitative polymerase chain reaction. RESULTS: Arno knockout mice are viable and fertile without any noted deficits. Arno wild type and knockout mice subjected to the OVA-induced model of rhinitis demonstrated an average of 314.5 and 153.8 EMBP+ cells per mm2 septal tissue, respectively (P < .05). Goblet cells per mm of basal lamina were assessed via Alcian blue and there was no statistically significant difference between Arno wild type and knockout mice. Ovalbumin-induced expression of interleukin-5 (IL-5) was significantly reduced in Arno knockout mice (P < .05). There was no statistically significant reduction in IL-4, IL-13, or eotaxin-1 expression. CONCLUSIONS: These data demonstrate that deletion of Arno reduces eosinophilic inflammation and IL-5 expression in an OVA-induced model of rhinitis.

Insights into patient preferences for elective surgery during the COVID-19 pandemic.

AIMS: To investigate factors that contribute to patient decisions regarding attendance for arthroplasty during the COVID-19 pandemic. METHODS: A postal questionnaire was distributed to patients on the waiting list for hip or knee arthroplasty in a single tertiary centre within the UK. Patient factors that may have influenced the decision to attend for arthroplasty, global quality of life (QoL) (EuroQol five-dimension three-level (EQ-5D-3L)), and joint-specific QoL (Oxford Hip or Knee Score) were assessed. Patients were asked at which 'COVID-alert' level they would be willing to attend an NHS and a "COVID-light" hospital for arthroplasty. Independent predictors were assessed using multivariate logistic regression. RESULTS: Of 540 distributed questionnaires, 400 (74.1%; 236 awaiting hip arthroplasty, 164 awaiting knee arthroplasty) complete responses were received and included. Less than half (48.2%) were willing to attend for hip or knee arthroplasty while a UK COVID-19 epidemic was in circulation (COVID-alert levels 3 to 5). Patients with worse joint-specific QoL had a preference to proceed with surgery at COVID-alert levels 3 to 5 compared to levels 1 and 2 (hip arthroplasty odds ratio (OR) 1.54 (95% confidence interval (CI) 1.45 to 1.63); knee arthroplasty OR 1.16 (1.07 to 1.26)). The odds of patients with worse joint-specific QoL being willing to attend for surgery at COVID-alert levels 3 to 5 increased further if surgery in a private, "COVID-light" hospital was available (hip arthroplasty OR 3.50 (95% CI 3.26 to 3.71); knee arthroplasty OR 1.41 (95% CI 1.29 to 1.53). CONCLUSION: Patient decisions surrounding elective surgery have been influenced by the global COVID-19 pandemic, highlighting the importance of patient involvement in ensuring optimized provision of elective surgery during these challenging times. Cite this article: Bone Jt Open 2021;2(4):261-270.

PI3Ka-Akt1-mediated Prdm4 induction in adipose tissue increases energy expenditure, inhibits weight gain, and improves insulin resistance in diet-induced obese mice.

Stimulation of white adipose tissue (WAT) browning is considered as a potential approach to treat obesity and metabolic diseases. Our previous studies have shown that phytochemical butein can stimulate WAT browning through induction of Prdm4 in adipocytes. Here, we investigated the effects of butein on diet-induced obesity and its underlying molecular mechanism. Treatment with butein prevented weight gains and improved metabolic profiles in diet-induced obese mice. Butein treatment groups also displayed higher body temperature, increased energy expenditure, and enhanced expression of thermogenic genes in adipose tissue. Butein also suppressed body weight gains and improved glucose and insulin tolerance in mice housed at thermoneutrality (30 °C). These effects were associated with adipose-selective induction of Prdm4, suggesting the role of Prdm4 in butein-mediated anti-obese effects. To directly assess the in vivo role of Prdm4, we generated aP2-Prdm4 transgenic mouse lines overexpressing Prdm4 in adipose tissues. Adipose-specific transgenic expression of Prdm4 recapitulated the butein's actions in stimulating energy expenditure, cold tolerance, and thermogenic gene expression, resulting in prevention of obesity and improvement of metabolism. Mechanistically, direct inhibition of PI3Kα activity followed by selective suppression of its downstream Akt1 mirrored butein's effect on Ucp1 expression and oxygen consumption. In addition, effects of butein were completely abolished in Akt1 KO mouse embryonic fibroblasts. Together, these studies demonstrate the role of butein in obesity and metabolic diseases, further highlighting that adipose PI3Kα-Akt1-Prdm4 axis is a regulator of energy expenditure.

Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy.

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.

ARF6 Is an Actionable Node that Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma.

Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as ß-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and ß-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.

Dietary Vitamin D and Its Metabolites Non-Genomically Stabilize the Endothelium.

Vitamin D is a known modulator of inflammation. Native dietary vitamin D3 is thought to be bio-inactive, and beneficial vitamin D3 effects are thought to be largely mediated by the metabolite 1,25(OH)2D3. Reduced serum levels of the most commonly measured precursor metabolite, 25(OH)D3, is linked to an increased risk of multiple inflammatory diseases, including: cardiovascular disease, arthritis, multiple sclerosis, and sepsis. Common to all of these diseases is the disruption of endothelial stability and an enhancement of vascular leak. We previously performed an unbiased chemical suppressor screen on a genetic model of vascular instability, and identified cholecalciferol (D3, dietary Vitamin D3) as a factor that had profound and immediate stabilizing and therapeutic effects in that model. In this manuscript we show that the presumed inactive sterol, D3, is actually a potent and general mediator of endothelial stability at physiologically relevant concentrations. We further demonstrate that this phenomenon is apparent in vitamin D3 metabolites 25(OH)D3 and 1,25(OH)2D3, and that the effects are independent of the canonical transcription-mediated vitamin D pathway. Our data suggests the presence of an alternative signaling modality by which D3 acts directly on endothelial cells to prevent vascular leak. The finding that D3 and its metabolites modulate endothelial stability may help explain the clinical correlations between low serum vitamin D levels and the many human diseases with well-described vascular dysfunction phenotypes.

Strategy for identifying repurposed drugs for the treatment of cerebral cavernous malformation.

BACKGROUND: Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans that has no approved nonsurgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. METHODS AND RESULTS: We developed an unbiased screening platform based on both cellular and animal models of loss of function of CCM2. Our discovery strategy consisted of 4 steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2, a secondary screen of functional changes in endothelial stability in these same cells, a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2, and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2100 known drugs and bioactive compounds and identified 2 candidates, cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide), for further study. Each drug decreased lesion burden in a mouse model of CCM vascular disease by ≈50%. CONCLUSIONS: By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.

Multiple roles for HOXA3 in regulating thymus and parathyroid differentiation and morphogenesis in mouse.

Hoxa3 was the first Hox gene to be mutated by gene targeting in mice and is required for the development of multiple endoderm and neural crest cell (NCC)-derived structures in the pharyngeal region. Previous studies have shown that the Hoxa3 null mutant lacks third pharyngeal pouch derivatives, the thymus and parathyroids by E18.5, and organ-specific markers are absent or downregulated during initial organogenesis. Our current analysis of the Hoxa3 null mutant shows that organ-specific domains did undergo initial patterning, but the location and timing of key regional markers within the pouch, including Tbx1, Bmp4 and Fgf8, were altered. Expression of the parathyroid marker Gcm2 was initiated but was quickly downregulated and differentiation failed; by contrast, thymus markers were delayed but achieved normal levels, concurrent with complete loss through apoptosis. To determine the cell type-specific roles of Hoxa3 in third pharyngeal pouch development, we analyzed tissue-specific mutants using endoderm and/or NCC-specific Cre drivers. Simultaneous deletion with both drivers resulted in athymia at E18.5, similar to the null. By contrast, the individual tissue-specific Hoxa3 deletions resulted in small, ectopic thymi, although each had a unique phenotype. Hoxa3 was primarily required in NCCs for morphogenesis. In endoderm, Hoxa3 temporally regulated initiation of the thymus program and was required in a cell-autonomous manner for parathyroid differentiation. Furthermore, Hoxa3 was required for survival of third pharyngeal pouch-derived organs, but expression in either tissue was sufficient for this function. These data show that Hoxa3 has multiple complex and tissue-specific functions during patterning, differentiation and morphogenesis of the thymus and parathyroids.

Mouse TBX3 mutants suggest novel molecular mechanisms for Ulnar-mammary syndrome.

The transcription factor TBX3 plays critical roles in development and TBX3 mutations in humans cause Ulnar-mammary syndrome. Efforts to understand how altered TBX3 dosage and function disrupt the development of numerous structures have been hampered by embryonic lethality of mice bearing presumed null alleles. We generated a novel conditional null allele of Tbx3: after Cre-mediated recombination, no mRNA or protein is detectable. In contrast, a putative null allele in which exons 1-3 are deleted produces a truncated protein that is abnormally located in the cytoplasm. Heterozygotes and homozygotes for this allele have different phenotypes than their counterparts bearing a true null allele. Our observations with these alleles in mice, and the different types of TBX3 mutations observed in human ulnar-mammary syndrome, suggest that not all mutations observed in humans generate functionally null alleles. The possibility that mechanisms in addition to TBX3 haploinsufficiency may cause UMS or other malformations merits investigation in the human UMS population.

Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1.

Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life. DOI:http://dx.doi.org/10.7554/eLife.00324.001.

The small GTPase ARF6 stimulates ß-catenin transcriptional activity during WNT5A-mediated melanoma invasion and metastasis.

ß-Catenin has a dual function in cells: fortifying cadherin-based adhesion at the plasma membrane and activating transcription in the nucleus. We found that in melanoma cells, WNT5A stimulated the disruption of N-cadherin and ß-catenin complexes by activating the guanosine triphosphatase adenosine diphosphate ribosylation factor 6 (ARF6). Binding of WNT5A to the Frizzled 4-LRP6 (low-density lipoprotein receptor-related protein 6) receptor complex activated ARF6, which liberated ß-catenin from N-cadherin, thus increasing the pool of free ß-catenin, enhancing ß-catenin-mediated transcription, and stimulating invasion. In contrast to WNT5A, the guidance cue SLIT2 and its receptor ROBO1 inhibited ARF6 activation and, accordingly, stabilized the interaction of N-cadherin with ß-catenin and reduced transcription and invasion. Thus, ARF6 integrated competing signals in melanoma cells, thereby enabling plasticity in the response to external cues. Moreover, small-molecule inhibition of ARF6 stabilized adherens junctions, blocked ß-catenin signaling and invasiveness of melanoma cells in culture, and reduced spontaneous pulmonary metastasis in mice, suggesting that targeting ARF6 may provide a means of inhibiting WNT/ß-catenin signaling in cancer.

Interleukin receptor activates a MYD88-ARNO-ARF6 cascade to disrupt vascular stability.

The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1ß (IL-1ß), which in turn activate a well-described, myeloid-differentiation factor 88 (MYD88)-mediated, nuclear factor-κB (NF-κB)-dependent transcriptional pathway that results in inflammatory-cell activation and recruitment. Endothelial cells, which usually serve as a barrier to the movement of inflammatory cells out of the blood and into tissue, are also critical mediators of the inflammatory response. Paradoxically, the cytokines vital to a successful immune defence also have disruptive effects on endothelial cell-cell interactions and can trigger degradation of barrier function and dissociation of tissue architecture. The mechanism of this barrier dissolution and its relationship to the canonical NF-κB pathway remain poorly defined. Here we show that the direct, immediate and disruptive effects of IL-1ß on endothelial stability in a human in vitro cell model are NF-κB independent and are instead the result of signalling through the small GTPase ADP-ribosylation factor 6 (ARF6) and its activator ARF nucleotide binding site opener (ARNO; also known as CYTH2). Moreover, we show that ARNO binds directly to the adaptor protein MYD88, and thus propose MYD88-ARNO-ARF6 as a proximal IL-1ß signalling pathway distinct from that mediated by NF-κB. Finally, we show that SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors such as ARNO, enhances vascular stability and significantly improves outcomes in animal models of inflammatory arthritis and acute inflammation.

Cytochrome P450 2B diversity and dietary novelty in the herbivorous, desert woodrat (Neotoma lepida).

Detoxification enzymes play a key role in plant-herbivore interactions, contributing to the on-going evolution of ecosystem functional diversity. Mammalian detoxification systems have been well studied by the medical and pharmacological industries to understand human drug metabolism; however, little is known of the mechanisms employed by wild herbivores to metabolize toxic plant secondary compounds. Using a wild rodent herbivore, the desert woodrat (Neotoma lepida), we investigated genomic structural variation, sequence variability, and expression patterns in a multigene subfamily involved in xenobiotic metabolism, cytochrome P450 2B (CYP2B). We hypothesized that differences in CYP2B expression and sequence diversity could explain differential abilities of woodrat populations to consume native plant toxins. Woodrats from two distinct populations were fed diets supplemented with either juniper (Juniperus osteosperma) or creosote bush (Larrea tridentata), plants consumed by woodrats in their respective desert habitats. We used Southern blot and quantitative PCR to determine that the genomic copy number of CYP2B in both populations was equivalent, and similar in number to known rodent copy number. We compared CYP2B expression patterns and sequence diversity using cloned hepatic CYP2B cDNA. The resulting sequences were very diverse, and clustered into four major clades by amino acid similarity. Sequences from the experimental treatments were distributed non-randomly across a CYP2B tree, indicating unique expression patterns from woodrats on different diets and from different habitats. Furthermore, within each major CYP2B clade, sequences shared a unique combination of amino acid residues at 13 sites throughout the protein known to be important for CYP2B enzyme function, implying differences in the function of each major CYP2B variant. This work is the most comprehensive investigation of the genetic diversity of a detoxification enzyme subfamily in a wild mammalian herbivore, and contributes an initial genetic framework to our understanding of how a wild herbivore responds to critical changes in its diet.

Netrin ligands and receptors: lessons from neurons to the endothelium.

Netrins were initially identified as secreted ligands regulating axon guidance and migration through interaction with canonical receptors. Netrins were then shown to be necessary for development of a range of tissues, including lung, mammary gland, and the vasculature. While new netrin receptors, as well as alternative ligands for classical netrin receptors, were described in the neuronal and epithelial fields, there was a singular focus on canonical netrin receptors in the vascular system, leading to controversy on netrin function and the nature of receptor-mediated netrin signaling in the endothelium. Here, we summarize the current state of knowledge on netrin ligands and receptors and discuss questions, controversies, and perspectives surrounding netrin functions and receptor identity in the vasculature.

Lethal arrhythmias in Tbx3-deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis.

TBX3 is critical for human development: mutations in TBX3 cause congenital anomalies in patients with ulnar-mammary syndrome. Data from mice and humans suggest multiple roles for Tbx3 in development and function of the cardiac conduction system. The mechanisms underlying the functional development, maturation, and maintenance of the conduction system are not well understood. We tested the requirements for Tbx3 in these processes. We generated a unique series of Tbx3 hypomorphic and conditional mouse mutants with varying levels and locations of Tbx3 activity within the heart, and developed techniques for evaluating in vivo embryonic conduction system function. Disruption of Tbx3 function in different regions of the developing heart causes discrete phenotypes and lethal arrhythmias: sinus pauses and bradycardia indicate sinoatrial node dysfunction, whereas preexcitation and atrioventricular block reveal abnormalities in the atrioventricular junction. Surviving Tbx3 mutants are at increased risk for sudden death. Arrhythmias induced by knockdown of Tbx3 in adults reveal its requirement for conduction system homeostasis. Arrhythmias in Tbx3-deficient embryos are accompanied by disrupted expression of multiple ion channels despite preserved expression of previously described conduction system markers. These findings indicate that Tbx3 is required for the conduction system to establish and maintain its correct molecular identity and functional properties. In conclusion, Tbx3 is required for the functional development, maturation, and homeostasis of the conduction system in a highly dosage-sensitive manner. TBX3 and its regulatory targets merit investigation as candidates for human arrhythmias.

Netrin-4 activates endothelial integrin {alpha}6{beta}1.

RATIONALE: Netrin-4 regulates vascular development. Identity of netrin-4 endothelial receptor and its subsequent cell functions is controversial. We previously demonstrated that the inhibition of netrin-1 canonical receptors, Unc5B and neogenin, expressed by lymphatic endothelial cells, do not suppress netrin-4-induced cell signaling and functions. Netrin family members were shown to signal through a range of receptors, including integrins (such as α3ß1, α6ß1, and α6ß4) in nonendothelial cells. OBJECTIVE: We tested whether integrins are netrin-4 receptors in the endothelium. METHODS AND RESULTS: The α6ß1 integrin is expressed by endothelial cells, and binds netrin-4 in a dose-dependent manner. Inhibition of α6 or ß1 integrin subunits suppresses netrin-4-induced endothelial cell migration, adhesion, and focal adhesion contact. Netrin-4-stimulated phosphorylation of Src kinase family, effectors of endothelial cell migration, is also abolished by α6 or ß1 inhibition. Finally, netrin-4 and α6ß1 integrin expression colocalize in mouse embryonic, intestine, and tumor vasculature. CONCLUSIONS: The α6ß1 integrin is a netrin-4 receptor in lymphatic endothelium and consequently represents a potential target to inhibit netrin-4-induced metastatic dissemination.

Techniques for cartilage repair in chondral and osteochondral defects of the knee.

Many techniques are currently used in an attempt to regenerate cartilage surfaces in the presence of a chondral or osteochondral defect. Clinical results have been mixed and no single treatment has emerged as being superior. This article reviews the techniques previously and currently being used and evidence to support their use.

Targeted deletion of the mouse Mitoferrin1 gene: from anemia to protoporphyria.

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show that the activity of mitoferrin1 is required to manage an increase in heme synthesis. The data also show that alterations in heme synthesis within hepatocytes can lead to protoporphyria and hepatotoxicity.

Netrin-4 induces lymphangiogenesis in vivo.

Netrin-4, a laminin-related secreted protein is an axon guidance cue recently shown essential outside of the nervous system, regulating mammary and lung morphogenesis as well as blood vascular development. Here, we show that Netrin-4, at physiologic doses, induces proliferation, migration, adhesion, tube formation and survival of human lymphatic endothelial cells in vitro comparable to well-characterized lymphangiogenic factors fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF), vascular endothelial growth factor-A (VEGF-A), and vascular endothelial growth factor-C (VEGF-C). Netrin-4 stimulates phosphorylation of intracellular signaling components Akt, Erk and S6, and their specific inhibition antagonizes Netrin-4-induced proliferation. Although Netrin receptors Unc5B and neogenin, are expressed by human lymphatic endothelial cells, suppression of either or both does not suppress Netrin-4-promoted in vitro effects. In vivo, Netrin-4 induces growth of lymphatic and blood vessels in the skin of transgenic mice and in breast tumors. Its overexpression in human and mouse mammary carcinoma cancer cells leads to enhanced metastasis. Finally, Netrin-4 stimulates in vitro and in vivo lymphatic permeability by activating small GTPases and Src family kinases/FAK, and down-regulating tight junction proteins. Together, these data provide evidence that Netrin-4 is a lymphangiogenic factor contributing to tumor dissemination and represents a potential target to inhibit metastasis formation.