Activation of NFAT by HGF and IGF-1 via ARF6 and its effector ASAP1 promotes uveal melanoma metastasis.

Preventing or effectively treating metastatic uveal melanoma (UM) is critical because it occurs in about half of patients and confers a very poor prognosis. There is emerging evidence that hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1) promote metastasis and contribute to the striking metastatic hepatotropism observed in UM metastasis. However, the molecular mechanisms by which HGF and IGF-1 promote UM liver metastasis have not been elucidated. ASAP1, which acts as an effector for the small GTPase ARF6, is highly expressed in the subset of uveal melanomas most likely to metastasize. Here, we found that HGF and IGF-1 hyperactivate ARF6, leading to its interaction with ASAP1, which then acts as an effector to induce nuclear localization and transcriptional activity of NFAT1. Inhibition of any component of this pathway impairs cellular invasiveness. Additionally, knocking down ASAP1 or inhibiting NFAT signaling reduces metastasis in a xenograft mouse model of UM. The discovery of this signaling pathway represents not only an advancement in our understanding of the biology of uveal melanoma metastasis but also identifies a novel pathway that could be targeted to treat or prevent metastatic uveal melanoma.

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.

Low-Cost Fluorescence Sensor for Ammonia Measurement in Livestock Houses.

Measurements of ammonia with inexpensive and reliable sensors are necessary to obtain information about e.g., ammonia emissions. The concentration information is needed for mitigation technologies and documentation of existing technologies in agriculture. A flow-based fluorescence sensor to measure ammonia gas was developed. The automated sensor is robust, flexible and made from inexpensive components. Ammonia is transferred to water in a miniaturized scrubber with high transfer efficiency (>99%) and reacts with o-phthalaldehyde and sulfite (pH 11) to form a fluorescent adduct, which is detected with a photodiode. Laboratory calibrations with standard gas show good linearity over a dynamic range from 0.03 to 14 ppm, and the detection limit of the analyzer based on three-times the standard deviation of blank noise was approximately 10 ppb. The sampling frequency is 0.1 to 10 s, which can easily be changed through serial commands along with UV LED current and filter length. Parallel measurements with a cavity ring-down spectroscopy analyzer in a pig house show good agreement (R2 = 0.99). The fluorescence sensor has the potential to provide ammonia gas measurements in an agricultural environment with high time resolution and linearity over a broad range of concentrations.

Modifiable Lifestyle Recommendations and Mortality in Denmark: A Cohort Study.

INTRODUCTION: Modifiable lifestyle behaviors represent a central target for public health interventions. This study investigates the association between adherence to 4 modifiable lifestyle recommendations and all-cause, cancer, or cardiovascular disease mortality. METHODS: Investigators used data from the Danish Diet, Cancer and Health cohort (1993-2013; N=54,276). Lifestyle recommendations included smoking (never smoking), diet (adherence to 6 national food-based dietary guidelines), alcohol consumption (≤7 units per week for women and ≤14 units per week for men), and physical activity (≥30 minutes per day of moderate-to-vigorous leisure-time physical activity). Pseudo-values were used to estimate the adjusted risk differences and 95% CIs for all-cause, cancer, or cardiovascular disease mortality. Data were analyzed in 2019-2020. RESULTS: A total of 8,860 participants died during a median follow-up of 17.0 years. Adherence to all modifiable lifestyle recommendations was associated with an 18.46% (95% CI= -20.52%, -16.41%) lower absolute risk of all-cause mortality than no adherence. Never smokers had a 13.19% (95% CI= -13.95%, -12.44%) lower risk, those adhering to dietary guidelines (diet score ≥5) had a 7.52% (95% CI= -8.89%, -6.14%) lower risk, and those adhering to recommended levels of alcohol (2.11%, 95% CI= -2.75%, -1.48%) and physical activity (1.58%, 95% CI= -2.20%, -1.00%) had a lower risk than those who did not adhere. Stronger associations were observed in men than in women and in older than in middle-aged participants. CONCLUSIONS: Findings suggest that adherence to modifiable lifestyle recommendations is associated with a lower risk of mortality from all causes, cancer, and cardiovascular disease, underlining the importance of supporting adherence to national guidelines for lifestyle recommendations.

The Small GTPase ARF6 Activates PI3K in Melanoma to Induce a Prometastatic State.

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients. SIGNIFICANCE: These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2892/F1.large.jpg.

Identification and Directed Development of Non-Organic Catalysts with Apparent Pan-Enzymatic Mimicry into Nanozymes for Efficient Prodrug Conversion.

Nanozymes, nanoparticles that mimic the natural activity of enzymes, are intriguing academically and are important in the context of the Origin of Life. However, current nanozymes offer mimicry of a narrow range of mammalian enzymes, near-exclusively performing redox reactions. We present an unexpected discovery of non-proteinaceous enzymes based on metals, metal oxides, 1D/2D-materials, and non-metallic nanomaterials. The specific novelty of these findings lies in the identification of nanozymes with apparent mimicry of diverse mammalian enzymes, including unique pan-glycosidases. Further novelty lies in the identification of the substrate scope for the lead candidates, specifically in the context of bioconversion of glucuronides, that is, human metabolites and privileged prodrugs in the field of enzyme-prodrug therapies. Lastly, nanozymes are employed for conversion of glucuronide prodrugs into marketed anti-inflammatory and antibacterial agents, as well as "nanozyme prodrug therapy" to mediate antibacterial measures.

Macromolecular prodrugs of ribavirin: Polymer backbone defines blood safety, drug release, and efficacy of anti-inflammatory effects.

Macromolecular (pro)drugs hold much promise as broad-spectrum antiviral agents as either microbicides or carriers for intracellular delivery of antiviral drugs. Intriguing opportunity exists in combining the two modes of antiviral activity in the same polymer structure such that the same polymer acts as a microbicide and also serves to deliver the conjugated drug (ribavirin) into the cells. We explore this opportunity in detail and focus on the polymer backbone as a decisive constituent of such formulations. Fourteen polyanions (polycarboxylates, polyphosphates and polyphosphonates, and polysulfonates) were analyzed for blood pro/anti coagulation effects, albumin binding and albumin aggregation, inhibitory activity on polymerases, cytotoxicity, and anti-inflammatory activity in stimulated macrophages. Ribavirin containing monomers were designed to accommodate the synthesis of macromolecular prodrugs with disulfide-exchange triggered drug release. Kinetics of drug release was fast in all cases however enhanced hydrophobicity of the polymer significantly slowed release of ribavirin. Results of this study present a comprehensive view on polyanions as backbone for macromolecular prodrugs of ribavirin as broad-spectrum antiviral agents.

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.

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.

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.

ARF6 inhibition stabilizes the vasculature and enhances survival during endotoxic shock.

The vascular endothelium responds to infection by destabilizing endothelial cell-cell junctions to allow fluid and cells to pass into peripheral tissues, facilitating clearance of infection and tissue repair. During sepsis, endotoxin and other proinflammatory molecules induce excessive vascular leak, which can cause organ dysfunction, shock, and death. Current therapies for sepsis are limited to antibiotics and supportive care, which are often insufficient to reduce morbidity and prevent mortality. Previous attempts at blocking inflammatory cytokine responses in humans proved ineffective at reducing the pathologies associated with sepsis, highlighting the need for a new therapeutic strategy. The small GTPase ARF6 is activated by a MyD88-ARNO interaction to induce vascular leak through disruption of endothelial adherens junctions. In this study, we show that the MyD88-ARNO-ARF6-signaling axis is responsible for LPS-induced endothelial permeability and is a destabilizing convergence point used by multiple inflammatory cues. We also show that blocking ARF6 with a peptide construct of its N terminus is sufficient to reduce vascular leak and enhance survival during endotoxic shock, without inhibiting the host cytokine response. Our data highlight the therapeutic potential of blocking ARF6 and reducing vascular leak for the treatment of inflammatory conditions, such as endotoxemia.

Smooth muscle specific disruption of the endothelin-A receptor in mice reduces arterial pressure, and vascular reactivity and affects vascular development.

AIMS: The role of vascular smooth muscle endothelin A receptors (ETA) in development and normal physiology remains incompletely understood. To address this, mice were generated with smooth muscle-specific knockout (KO) of ETA. MAIN METHODS: Mice were homozygous for loxP-flanked exons 6-8 of the EDNRA gene (floxed) or were also hemizygous for a transgene expressing Cre recombinase under control of the smooth muscle-specific SM22 promoter (KO mice). KEY FINDINGS: Genotyping at 17 days postnatal yielded a 10:1 ratio of floxed:KO mice. Smooth muscle actin staining of embryos at day E10.5 revealed increased tortuosity in dorsal aortae while E12.5 embryos had mandibular, vascular and thymic abnormalities. Mice surviving to weaning developed and bred normally. ETA KO mice aged 2-3 months manifested EDNRA gene recombination in all organs tested. Aortas from KO mice had a >90% reduction in ETA mRNA content, but no differences in ET-1 or ETB mRNA levels. Addition of 0.01-100 nM ET-1 to isolated femoral arteries from floxed, but not KO, mice dose-dependently decreased vessel diameter (up to 80% reduction in the presence of ETB blockade). Intravenous infusion of ET-1 into floxed, but not KO, mice increased mean arterial pressure (MAP) (by ~10 mm Hg). Telemetric analysis revealed decreased MAP in KO mice (reduced by ~7-10 mm Hg) when fed a high salt diet. SIGNIFICANCE: Smooth muscle ETA is important for normal vascular, mandibular and thymic development and is involved in the maintenance of arterial pressure under physiological conditions.

Motivational interviewing: a part of the weight loss program for overweight and obese women prior to fertility treatment.

This is a retrospective study to investigate whether motivational interviewing increases weight loss among obese or overweight women prior to fertility treatment. Women with body mass index (BMI) > 30 kg/m(2) approaching the Fertility Clinic, Regional Hospital Skive, were given advice about diet and physical activity with the purpose of weight loss. In addition, they were asked if they wanted to receive motivational interviewing. Among other data, age, height and weight were obtained. Main outcomes were weight loss measured in kg and decrease in BMI. We studied 187 women: 110 received sessions of motivational interviewing (intervention group, n = 110), 64 received motivational support by phone or e-mail only and 13 women did not wish any motivational support (control group, n = 77). The mean weight loss and decrease in BMI was greater in the intervention group compared with the control group (9.3 kg versus 7.3 kg, difference p = 0.01, 3.3 kg/m(2) versus 2.6 kg/m(2), difference p = 0.02). The mean period of intervention was comparable in the two groups, 7.9 month and 7.3 month, respectively, (difference non significant: NS). The study indicates that motivational interviewing may be a valuable tool in weight loss programs for obese and overweight women prior to fertility treatment.

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.

Novel approach for endothelializing vascular devices: understanding and exploiting elastin-endothelial interactions.

Elastin is an essential component of arteries which provides structural integrity and instructs smooth muscle cells to adopt a quiescent state. Despite interaction of endothelial cells with elastin in the internal elastic lamina, the potential for exploiting this interaction therapeutically has not been explored in detail. In this study, we show that tropoelastin (a precursor of elastin) stimulates endothelial cell migration and adhesion more than smooth muscle cells. The biological activity of tropoelastin on endothelial cells is contained in the VGVAPG domain and in the carboxy-terminal 17-amino acids. We show that the effects of the carboxy-terminal 17 amino acids, but not those of VGVAPG, are mediated by integrin α(V)ß(3). We demonstrate that tropoelastin covalently linked to stainless steel disks promotes adhesion of endothelial progenitor cells and endothelial cells to the metal surfaces. The adherent cells on the tropoelastin-coated metal surfaces form monolayers that can withstand and respond to arterial shear stress. Because of the unique effects of tropoelastin on endothelial and smooth muscle cells, coating intravascular devices with tropoelastin may stimulate their endothelialization, inhibit smooth muscle hyperplasia, and improve device performance.

Vascular Robo4 restricts proangiogenic VEGF signaling in breast.

Formation of the vascular system within organs requires the balanced action of numerous positive and negative factors secreted by stromal and epithelial cells. Here, we used a genetic approach to determine the role of SLITs in regulating the growth and organization of blood vessels in the mammary gland. We demonstrate that vascularization of the gland is not affected by loss of Slit expression in the epithelial compartment. Instead, we identify a stromal source of SLIT, mural cells encircling blood vessels, and show that loss of Slit in the stroma leads to elevated blood vessel density and complexity. We examine candidate SLIT receptors, Robo1 and Robo4, and find that increased vessel angiogenesis is phenocopied by loss of endothelial-specific Robo4, as long as it is combined with the presence of an angiogenic stimulus such as preneoplasia or pregnancy. In contrast, loss of Robo1 does not affect blood vessel growth. The enhanced growth of blood vessels in Robo4(-/-) endothelium is due to activation of vascular endothelial growth factor (VEGF)-R2 signaling through the Src and FAK kinases. Thus, our studies present a genetic dissection of SLIT/ROBO signaling during organ development. We identify a stromal, rather than epithelial, source of SLITs that inhibits blood vessel growth by signaling through endothelial ROBO4 to down-regulate VEGF/VEGFR2 signaling.

Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza.

The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1beta and tumor necrosis factor-alpha, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host's immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host's response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1 influenza. Thus, enhancing the resilience of the host vascular system to the host's innate immune response may provide a therapeutic strategy for treating multiple infectious agents.