Targeted action to increase inclusion at the Wellcome Sanger Institute.

The Sanger Excellence Fellowship has been established to increase the representation of researchers with Black-heritage backgrounds at a leading research centre in the UK.

Apoferritin encapsulation of cysteine protease inhibitors for cathepsin L inhibition in cancer cells.

Cysteine proteases play a key role in tumorigenesis causing protein degradation and promoting invasive tumour growth. Cathepsin L is overexpressed in cancer cells and could provide a specific target for delivery of anticancer agents. We encapsulated novel dipeptidyl nitrile based cysteine protease inhibitors (Neq0551, Neq0554 and Neq0568) into biocompatible apoferritin (AFt) protein nanocages to achieve specific delivery to tumours and pH-induced drug release. AFt-encapsulated Neq0554 demonstrated ∼3-fold enhanced in vitro activity (GI50 = 79 µM) compared to naked agent against MiaPaCa-2 pancreatic carcinoma cells. Selectivity for cancer cells was confirmed by comparing their activity to non-tumourigenic human fibroblasts (GI50 > 200 µM). Transferrin receptor (TfR-1) expression, detected only in lysates prepared from carcinoma cells, may contribute to the cancer-selectivity. The G1 cell cycle arrest caused by AFt-Neq0554 resulting in cytostasis was corroborated by clonogenic assays. Superior and more persistent inhibition of cathepsin L up to 80% was achieved with AFt-encapsulated agent in HCT-116 cells following 6 h exposure to 50 µM agent. The selective anticancer activity of AFt-encapsulated cysteine protease inhibitor Neq0554 reported here warrants further preclinical in vivo evaluation.

A non-functional galanin receptor-2 in a multiple sclerosis patient.

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.

Molecular Pharmacology of VEGF-A Isoforms: Binding and Signalling at VEGFR2.

Vascular endothelial growth factor-A (VEGF-A) is a key mediator of angiogenesis, signalling via the class IV tyrosine kinase receptor family of VEGF Receptors (VEGFRs). Although VEGF-A ligands bind to both VEGFR1 and VEGFR2, they primarily signal via VEGFR2 leading to endothelial cell proliferation, survival, migration and vascular permeability. Distinct VEGF-A isoforms result from alternative splicing of the Vegfa gene at exon 8, resulting in VEGFxxxa or VEGFxxxb isoforms. Alternative splicing events at exons 5⁻7, in addition to recently identified posttranslational read-through events, produce VEGF-A isoforms that differ in their bioavailability and interaction with the co-receptor Neuropilin-1. This review explores the molecular pharmacology of VEGF-A isoforms at VEGFR2 in respect to ligand binding and downstream signalling. To understand how VEGF-A isoforms have distinct signalling despite similar affinities for VEGFR2, this review re-evaluates the typical classification of these isoforms relative to the prototypical, “pro-angiogenic” VEGF165a. We also examine the molecular mechanisms underpinning the regulation of VEGF-A isoform signalling and the importance of interactions with other membrane and extracellular matrix proteins. As approved therapeutics targeting the VEGF-A/VEGFR signalling axis largely lack long-term efficacy, understanding these isoform-specific mechanisms could aid future drug discovery efforts targeting VEGF receptor pharmacology.

A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells.

Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A3 receptor (A3AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A1 receptor, and ß1 and ß2 adrenoceptors (ß1AR and ß2AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A3AR for a range of classical adenosine receptor antagonists were consistent with A3AR pharmacology and correlated well (R2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the ß1AR was potently inhibited by low concentrations of the ß1-selective antagonist CGP 20712A (pKi 9.68) but not by the ß2-selective antagonist ICI 118551(pKi 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the ß2AR this affinity order was reversed with ICI 118551 showing the highest affinity (pKi 8.73) and CGP20712A (pKi 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A3AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A3AR (pKi ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A3AR. These were K114 (pKi 6.43), retinoic acid p-hydroxyanilide (pKi 6.13) and SU 6556 (pKi 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A3AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors.

Redox modulation of FAK controls melanoma survival--role of NOX4.

Studies have demonstrated that reactive oxygen species (ROS) generated by NADPH oxidase are essential for melanoma proliferation and survival. However, the mechanisms by which NADPH oxidase regulates these effects are still unclear. In this work, we investigate the role of NADPH oxidase-derived ROS in the signaling events that coordinate melanoma cell survival. Using the highly metastatic human melanoma cell line MV3, we observed that pharmacological NADPH oxidase inhibition reduced melanoma viability and induced dramatic cellular shape changes. These effects were accompanied by actin cytoskeleton rearrangement, diminished FAKY397 phosphorylation, and decrease of FAK-actin and FAK-cSrc association, indicating disassembly of focal adhesion processes, a phenomenon that often results in anoikis. Accordingly, NADPH oxidase inhibition also enhanced hypodiploid DNA content, and caspase-3 activation, suggesting activation of the apoptotic machinery. NOX4 is likely to be involved in these effects, since silencing of NOX4 significantly inhibited basal ROS production, reduced FAKY397 phosphorylation and decreased tumor cell viability. Altogether, the results suggest that intracellular ROS generated by the NADPH oxidase, most likely NOX4, transmits cell survival signals on melanoma cells through the FAK pathway, maintaining adhesion contacts and cell viability.

Essential roles of PKA, iNOS, CD95/CD95L, and terminal caspases in suppression of eosinopoiesis by PGE2 and other cAMP-elevating agents.

Up- and downregulation of eosinopoiesis control pulmonary eosinophilia in human asthma. In mice, eosinopoiesis is suppressed in vitro by prostaglandin E2 (PGE2) and in vivo by diethylcarbamazine, through a proapoptotic mechanism sequentially requiring inducible NO synthase (iNOS) and the ligand for death receptor CD95 (CD95L). We examined the roles of iNOS, cAMP-mediated signaling, caspases, and CD95L/CD95 in suppression of eosinopoiesis by PGE2 and other agents signaling through cAMP. Bone-marrow collected from BALB/c mice, or from iNOS-, CD95-, or CD95L-deficient mutants (and wild-type controls), was cultured with interleukin-5 (IL-5), alone or associated with PGE2, cAMP-inducing/mimetic agents, caspase inhibitor zVAD-fmk, iNOS inhibitor aminoguanidine, or combinations thereof, and eosinopoiesis was evaluated at various times. PGE2, added up to 24 hours of culture, dose-dependently suppressed eosinopoiesis, by inducing apoptosis. This effect was (a) paralleled by induction of iNOS in eosinophils; (b) duplicated by sodium nitroprusside, isoproterenol, and cAMP-inducing/mimetic agents; (c) prevented by protein kinase A inhibition. NO was produced through iNOS by dibutyryl-cAMP-stimulated bone-marrow. Overall, PGE2 and isoproterenol shared a requirement for four effector elements (iNOS, CD95L, CD95, and terminal caspases), which together define a pathway targeted by several soluble up- and downmodulators of eosinopoiesis, including drugs, mediators of inflammation, and cytokines.

Proinflammatory responses of heme in alveolar macrophages: repercussion in lung hemorrhagic episodes.

Clinical and experimental observations have supported the notion that free heme released during hemorrhagic and hemolytic episodes may have a major role in lung inflammation. With alveolar macrophages (AM) being the main line of defense in lung environments, the influence of free heme on AM activity and function was investigated. We observed that heme in a concentration range found during hemolytic episodes (3-30 µM) elicits AM to present a proinflammatory profile, stimulating reactive oxygen species (ROS) and nitric oxide (NO) generation and inducing IL-1ß, IL-6, and IL-10 secretion. ROS production is NADPH oxidase-dependent, being inhibited by DPI and apocynin, and involves p47 subunit phosphorylation. Furthermore, heme induces NF- κB nuclear translocation, iNOS, and also HO-1 expression. Moreover, AM stimulated with free heme show enhanced phagocytic and bactericidal activities. Taken together, the data support a dual role for heme in the inflammatory response associated with lung hemorrhage, acting as a proinflammatory molecule that can either act as both an adjuvant of the innate immunity and as an amplifier of the inflammatory response, leading tissue injury. The understanding of heme effects on pulmonary inflammatory processes can lead to the development of new strategies to ameliorate tissue damage associated with hemorrhagic episodes.

Heme modulates intestinal epithelial cell activation: involvement of NADPHox-derived ROS signaling.

In many gut chronic inflammatory conditions, intestinal epithelium (IE) is deprived of the protection of the mucus secreted by IE-specialized cells. In these events, bleeding and subsequent lysis of erythrocytes are common. This may lead to the release of high amounts of heme in the intestinal lumen, which interacts with IE. Previous works from our group have shown that heme itself is a proinflammatory molecule, activating a number of phlogistic signaling events in a nicotinamide adenine dinucleotide phosphate oxidase (NADPHox)-dependent manner. In this study, we aim to evaluate the effects of heme upon a well-established nontransformed small intestine epithelial cell lineage (IEC 6). Our results show that free heme evokes intracellular reactive oxygen species (ROS) production by IEC 6 cells, which is inhibited both by pharmacological inhibition with diphenyleneiodonium (10 µM), a NADPHox inhibitor, and small interfering RNA-mediated suppression of NOX1, a constitutive NADPHox isoform present in intestinal epithelial cells. Focal adhesion kinase phosphorylation and actin cytoskeleton polymerization are also induced by heme in a NADPHox-dependent manner. Heme increases monolayer permeability and redistributes key modulators of cell-cell adhesion as zona occludens-1 and E-cadherin proteins via NADPHox signaling. Heme promotes IEC 6 cell migration and proliferation, phenomena also regulated by NADPHox-derived ROS. Heme, in NADPHox-activating concentrations, is able to induce mRNA expression of IL-6, a cytokine implicated in inflammatory and tumorigenic responses. These data indicate a prominent role for heme-derived signaling in the pathophysiology of intestinal mucosa dysfunction and address an important role of NADPHox activity on the pathogenesis of intestinal inflammatory conditions.

Leukotriene B(4) inhibits neutrophil apoptosis via NADPH oxidase activity: redox control of NF-κB pathway and mitochondrial stability.

Leukotriene B(4), an arachidonic acid-derived lipid mediator, is a known proinflammatory agent that has a direct effect upon neutrophil physiology, inducing reactive oxygen species generation by the NADPH oxidase complex and impairing neutrophil spontaneous apoptosis, which in turn may corroborate to the onset of chronic inflammation. Despite those facts, a direct link between inhibition of neutrophil spontaneous apoptosis and NADPH oxidase activation by leukotriene B(4) has not been addressed so far. In this study, we aim to elucidate the putative role of NADPH oxidase-derived reactive oxygen species in leukotriene B(4)-induced anti-apoptotic effect. Our results indicate that NADPH oxidase-derived reactive oxygen species are critical to leukotriene B(4) pro-survival effect on neutrophils. This effect also relies on redox modulation of nuclear factor kappaB signaling pathway. We have also observed that LTB(4)-induced Bad degradation and mitochondrial stability require NADPH oxidase activity. All together, our results strongly suggest that LTB(4)-induced anti-apoptotic effect in neutrophils occurs in a reactive oxygen species-dependent manner. We do believe that a better knowledge of the molecular mechanisms underlying neutrophil spontaneous apoptosis may contribute to the development of more successful strategies to control chronic inflammatory conditions such as rheumatoid arthritis.

NADPH oxidase activity: In the crossroad of neutrophil life and death.

Neutrophils are terminally differentiated leukocytes, specialized in detecting and annihilating possible pathogens. For this function, neutrophils contain a number of cytotoxic systems, which can both kill the intruder or promote extensive tissue injury. The most stereotyped neutrophil cytotoxic mechanism is the extracellular and intra-phagosomal production of high amounts of superoxide (O2-) and other reactive oxygen species (ROS) via the activation of the complex NADPH oxidase (NADPHox). It has been proposed that the short neutrophil lifespan would be a mechanism of counter-regulating the indiscriminate release of its cytotoxic content, as well as aborting the excessive production of ROS. Studies performed in the last decades point out the role of NADPHox activity as one of the major systems involved in the up-regulation of neutrophil apoptosis. However, a growing number of evidence suggests that NADPHox-derived ROS are involved in the activation of signaling pathways that may lead to increased neutrophil survival. In this review, we evaluate the implication of NADPHox activity in the control of neutrophil's life and death, highlighting the signaling pathways modulated by NADPHox-derived ROS.

Aspirin-triggered lipoxin A4 blocks reactive oxygen species generation in endothelial cells: a novel antioxidative mechanism.

Lipoxins and their aspirin-triggered carbon-15 epimers have emerged as mediators of key events in endogenous anti-inflammation and resolution. However, the implication of these novel lipid mediators on cardiovascular diseases such as hypertension, atherosclerosis, and heart failure has not been investigated. One of the major features shared by these pathological conditions is the increased production of reactive oxygen species (ROS) generated by vascular NAD(P)H oxidase activation. In this study, we have examined whether an aspirin-triggered lipoxin A (4) analog (ATL-1) modulates ROS generation in endothelial cells (EC). Pre-treatment of EC with ATL-1 (1 - 100 nM) completely blocked ROS production triggered by different agents, as assessed by dihydrorhodamine 123 and hydroethidine. Furthermore, ATL-1 inhibited the phosphorylation and translocation of the cytosplamic NAD(P)H oxidase subunit p47 (phox) to the cell membrane as well as NAD(P)H oxidase activity. Western blot and immunofluorescence microscopy analyses showed that ATL-1 (100 nM) impaired the redox-sensitive activation of the transcriptional factor NF- kappaB, a critical step in several events associated to vascular pathologies. These results demonstrate that ATL-1 suppresses NAD(P)H oxidase-mediated ROS generation in EC, strongly indicating that lipoxins may play a protective role against the development and progression of cardiovascular diseases.

NADPH oxidase-derived ROS: key modulators of heme-induced mitochondrial stability in human neutrophils.

Heme is a proinflammatory molecule able to cause a profound delay of constitutive apoptosis of human neutrophils, an effect that likely contributes to chronic inflammation associated with hemolytic diseases. Herein we show that heme-induced delay of neutrophil apoptosis correlates with the prevention of mitochondrial potential (Deltapsi(m)) dissipation by a mechanism dependent on NADPH oxidase (NADPHox)-generated reactive oxygen species (ROS) and NF-kappaB. Deltapsi(m) maintenance is accompanied by inhibition of Bax insertion into mitochondria and by a decrease in the Bad/Bcl-X(L) ratio. Heme induces Bad degradation in a completely ROS-dependent manner, as well as Bcl-X(L) synthesis, a phenomenon that also requires NF-kappaB activation. These data indicate that heme-induced preservation of mitochondrial integrity is a critical checkpoint controlled by NADPH oxidase generated-ROS and redox-sensitive NF-kappaB activation.

Heme and innate immunity: new insights for an old molecule

Hemolytic episodes such as sickle cell disease, malaria and ischemia-reperfusion occurrence are often associated to the statement of an inflammatory response which may develop or not to a chronic inflammatory status. Although these pathological states are triggered by distinct etiological agents, all of them are associated to high levels of free heme in circulation. In this review, we aim to focus the very recent achievements that have led to the statement of free heme as a proinflammatory molecule, which may play a central role during the onset and/or persistance of inflammation during these pathologies.

Heme and innate immunity: new insights for an old molecule

Hemolytic episodes such as sickle cell disease, malaria and ischemia-reperfusion occurrence are often associated to the statement of an inflammatory response which may develop or not to a chronic inflammatory status. Although these pathological states are triggered by distinct etiological agents, all of them are associated to high levels of free heme in circulation. In this review, we aim to focus the very recent achievements that have led to the statement of free heme as a proinflammatory molecule, which may play a central role during the onset and/or persistance of inflammation during these pathologies.

Heme and innate immunity: new insights for an old molecule.

Hemolytic episodes such as sickle cell disease, malaria and ischemia-reperfusion occurrence are often associated to the statement of an inflammatory response which may develop or not to a chronic inflammatory status. Although these pathological states are triggered by distinct etiological agents, all of them are associated to high levels of free heme in circulation. In this review, we aim to focus the very recent achievements that have led to the statement of free heme as a proinflammatory molecule, which may play a central role during the onset and/or persistence of inflammation during these pathologies.

Heme inhibits human neutrophil apoptosis: involvement of phosphoinositide 3-kinase, MAPK, and NF-kappaB.

High levels of free heme are found in pathological states of increased hemolysis, such as sickle cell disease, malaria, and ischemia reperfusion. The hemolytic events are often associated with an inflammatory response that usually turns into chronic inflammation. We recently reported that heme is a proinflammatory molecule, able to induce neutrophil migration, reactive oxygen species generation, and IL-8 expression. In this study, we show that heme (1-50 microM) delays human neutrophil spontaneous apoptosis in vitro. This effect requires heme oxygenase activity, and depends on reactive oxygen species production and on de novo protein synthesis. Inhibition of ERK and PI3K pathways abolished heme-protective effects upon human neutrophils, suggesting the involvement of the Ras/Raf/MAPK and PI3K pathway on this effect. Confirming the involvement of these pathways in the modulation of the antiapoptotic effect, heme induces Akt phosphorylation and ERK-2 nuclear translocation in neutrophils. Futhermore, inhibition of NF-kappa B translocation reversed heme antiapoptotic effect. NF-kappa B (p65 subunit) nuclear translocation and I kappa B degradation were also observed in heme-treated cells, indicating that free heme may regulate neutrophil life span modulating signaling pathways involved in cell survival. Our data suggest that free heme associated with hemolytic episodes might play an important role in the development of chronic inflammation by interfering with the longevity of neutrophils.

Neutrophil activation by heme: implications for inflammatory processes.

Heme, a ubiquitous iron-containing compound, is present in large amounts in many cells and is inherently dangerous, particularly when it escapes from intracellular sites. The release of heme from damaged cells and tissues is supposed to be higher in diseases such as malaria and hemolytic anemia or in trauma and hemorrhage. We investigated here the role of free ferriprotoporphyrin IX (hemin) as a proinflammatory molecule, with particular attention to its ability to activate neutrophil responses. Injecting hemin into the rat pleural cavity resulted in a dose-dependent migration of neutrophils, indicating that hemin is able to promote the recruitment of these cells in vivo. In vitro, hemin induced human neutrophil chemotaxis and cytoskeleton reorganization, as revealed by the increase of neutrophil actin polymerization. Exposure of human neutrophils to 3 microM hemin activated the expression of the chemokine interleukin-8, as demonstrated by quantitative reverse-transcription polymerase chain reaction, indicating a putative molecular mechanism by which hemin induces chemotaxis in vivo. Brief incubation of human neutrophils with micromolar concentrations of hemin (1-20 microM) triggered the oxidative burst, and the production of reactive oxygen species was directly proportional to the concentration of hemin added to the cells. Finally, we observed that human neutrophil protein kinase C was activated by hemin in vitro, with a K(1/2) of 5 microM. Taken together, these results suggest a role for hemin as a proinflammatory agent able to induce polymorphonuclear neutrophil activation in situations of clinical relevance, such as hemolysis or hemoglobinemia.