Defining a Water-Soluble Formulation of Arachidonic Acid as a Novel Ferroptosis Inducer in Cancer Cells.

Here, we describe GS-9, a novel water-soluble fatty acid-based formulation comprising L-lysine and arachidonic acid, that we have shown to induce ferroptosis. GS-9 forms vesicle-like structures in solution and mediates lipid peroxidation, as evidenced by increased C11-BODIPY fluorescence and an accumulation of toxic malondialdehyde, a downstream product of lipid peroxidation. Ferroptosis inhibitors counteracted GS-9-induced cell death, whereas caspase 3 and 7 or MLKL knock-out cell lines are resistant to GS-9-induced cell death, eliminating other cell death processes such as apoptosis and necroptosis as the mechanism of action of GS-9. We also demonstrate that through their role of sequestering fatty acids, lipid droplets play a protective role against GS-9-induced ferroptosis, as inhibition of lipid droplet biogenesis enhanced GS-9 cytotoxicity. In addition, Fatty Acid Transport Protein 2 was implicated in GS-9 uptake. Overall, this study identifies and characterises the mechanism of GS-9 as a ferroptosis inducer. This formulation of arachidonic acid offers a novel tool for investigating and manipulating ferroptosis in various cellular and anti-cancer contexts.

Heparanase promotes the onset and progression of atherosclerosis in apolipoprotein E gene knockout mice.

BACKGROUND AND AIMS: Atherosclerosis is the primary underlying cause of myocardial infarction and stroke, which are the major causes of death globally. Heparanase (Hpse) is a pro-inflammatory extracellular matrix degrading enzyme that has been implicated in atherogenesis. However, to date the precise roles of Hpse in atherosclerosis and its mechanisms of action are not well defined. This study aims to provide new insights into the contribution of Hpse in different stages of atherosclerosis in vivo. METHODS: We generated Hpse gene-deficient mice on the atherosclerosis-prone apolipoprotein E gene knockout (ApoE-/-) background to investigate the impact of Hpse gene deficiency on the initiation and progression of atherosclerosis after 6 and 14 weeks high-fat diet feeding, respectively. Atherosclerotic lesion development, blood serum profiles, lesion composition and aortic immune cell populations were evaluated. RESULTS: Hpse-deficient mice exhibited significantly reduced atherosclerotic lesion burden in the aortic sinus and aorta at both time-points, independent of changes in plasma cholesterol levels. A significant reduction in the necrotic core size and an increase in smooth muscle cell content were also observed in advanced atherosclerotic plaques of Hpse-deficient mice. Additionally, Hpse deficiency reduced circulating and aortic levels of VCAM-1 at the initiation and progression stages of disease and circulating MCP-1 levels in the initiation but not progression stage. Moreover, the aortic levels of total leukocytes and dendritic cells in Hpse-deficient ApoE-/- mice were significantly decreased compared to control ApoE-/-mice at both disease stages. CONCLUSIONS: This study identifies Hpse as a key pro-inflammatory enzyme driving the initiation and progression of atherosclerosis and highlighting the potential of Hpse inhibitors as novel anti-inflammatory treatments for cardiovascular disease.

The small molecule raptinal can simultaneously induce apoptosis and inhibit PANX1 activity.

Discovery of new small molecules that can activate distinct programmed cell death pathway is of significant interest as a research tool and for the development of novel therapeutics for pathological conditions such as cancer and infectious diseases. The small molecule raptinal was discovered as a pro-apoptotic compound that can rapidly trigger apoptosis by promoting the release of cytochrome c from the mitochondria and subsequently activating the intrinsic apoptotic pathway. As raptinal is very effective at inducing apoptosis in a variety of different cell types in vitro and in vivo, it has been used in many studies investigating cell death as well as the clearance of dying cells. While examining raptinal as an apoptosis inducer, we unexpectedly identified that in addition to its pro-apoptotic activities, raptinal can also inhibit the activity of caspase-activated Pannexin 1 (PANX1), a ubiquitously expressed transmembrane channel that regulates many cell death-associated processes. By implementing numerous biochemical, cell biological and electrophysiological approaches, we discovered that raptinal can simultaneously induce apoptosis and inhibit PANX1 activity. Surprisingly, raptinal was found to inhibit cleavage-activated PANX1 via a mechanism distinct to other well-described PANX1 inhibitors such as carbenoxolone and trovafloxacin. Furthermore, raptinal also interfered with PANX1-regulated apoptotic processes including the release of the 'find-me' signal ATP, the formation of apoptotic cell-derived extracellular vesicles, as well as NLRP3 inflammasome activation. Taken together, these data identify raptinal as the first compound that can simultaneously induce apoptosis and inhibit PANX1 channels. This has broad implications for the use of raptinal in cell death studies as well as in the development new PANX1 inhibitors.

Heparanase: A Novel Therapeutic Target for the Treatment of Atherosclerosis.

Cardiovascular disease (CVD) is the leading cause of death and disability worldwide, and its management places a huge burden on healthcare systems through hospitalisation and treatment. Atherosclerosis is a chronic inflammatory disease of the arterial wall resulting in the formation of lipid-rich, fibrotic plaques under the subendothelium and is a key contributor to the development of CVD. As such, a detailed understanding of the mechanisms involved in the development of atherosclerosis is urgently required for more effective disease treatment and prevention strategies. Heparanase is the only mammalian enzyme known to cleave heparan sulfate of heparan sulfate proteoglycans, which is a key component of the extracellular matrix and basement membrane. By cleaving heparan sulfate, heparanase contributes to the regulation of numerous physiological and pathological processes such as wound healing, inflammation, tumour angiogenesis, and cell migration. Recent evidence suggests a multifactorial role for heparanase in atherosclerosis by promoting underlying inflammatory processes giving rise to plaque formation, as well as regulating lesion stability. This review provides an up-to-date overview of the role of heparanase in physiological and pathological processes with a focus on the emerging role of the enzyme in atherosclerosis.

Human ß-Defensin 2 (HBD-2) Displays Oncolytic Activity but Does Not Affect Tumour Cell Migration.

Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. Therefore, HDPs have been explored as promising anticancer therapeutics. Human ß-defensin 2 (HBD-2) represents a prominent member of human HDPs, being well-characterised for its potent pathogen-killing, wound-healing, cytokine-inducing and leukocyte-chemoattracting functions. However, its anticancer effects remain largely unknown. Recently, we demonstrated that HBD-2 binds strongly to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), a key mediator of defensin-induced cell death and an instructional messenger during cell migration. Hence, in this study, we sought to investigate the lytic and anti-migratory effects of HBD-2 on tumour cells. Using various cell biological assays and confocal microscopy, we showed that HBD-2 killed tumour cells via acute lytic cell death rather than apoptosis. In addition, our data suggested that, despite the reported PI(4,5)P2 interaction, HBD-2 does not affect cytoskeletal-dependent tumour cell migration. Together, our findings provide further insights into defensin biology and informs future defensin-based drug development.

Gift bags from the sentinel cells of the immune system: The diverse role of dendritic cell-derived extracellular vesicles.

Dendritic cells (DCs) are professional APCs of the immune system that continuously sample their environment and function to stimulate an adaptive immune response by initiating Ag-specific immunity or tolerance. Extracellular vesicles (EVs), small membrane-bound structures, are released from DCs and have been discovered to harbor functional peptide-MHC complexes, T cell costimulatory molecules, and other molecules essential for Ag presentation, immune cell regulation, and stimulating immune responses. As such, DC-derived EVs are being explored as potential immunotherapeutic agents. DC-derived EVs have also been implicated to function as a trafficking mechanism of infectious particles aiding viral propagation. This review will explore the unique features that enable DC-derived EVs to regulate immune responses and interact with recipient cells, their roles within Ag-presentation and disease settings, as well as speculating on a potential immunological role of apoptotic DC-derived EVs.

Construction of a Highly Sensitive Thiol-Reactive AIEgen-Peptide Conjugate for Monitoring Protein Unfolding and Aggregation in Cells.

Impairment of the protein quality control network leads to the accumulation of unfolded and aggregated proteins. Direct detection of unfolded protein accumulation in the cells may provide the possibility for early diagnosis of neurodegenerative diseases. Here a new platform based on a peptide-conjugated thiol-reactive aggregation-induced emission fluorogen (AIEgen), named MI-BTD-P (or D1), for labeling and tracking unfolded proteins in cells is reported. In vitro experiments with model proteins show that the non-fluorescent D1 only becomes highly fluorescent when reacted with the thiol group of free cysteine (Cys) residues on unfolded proteins but not glutathione or folded proteins with buried or surface exposed Cys. When the labeled unfolded proteins form aggregates, D1 fluorescence intensity is further increased, and fluorescence lifetime is prolonged. D1 is then used to measure unfolded protein loads in cells by flow cytometry and track the aggregate formation of the D1 labeled unfolded proteins using confocal microscopy. In combination with fluorescence lifetime imaging technique, the proteome at different folding statuses can be better differentiated, demonstrating the versatility of this new platform. The rational design of D1 demonstrates the outlook of incorporation of diverse functional groups to achieve maximal sensitivity and selectivity in biological samples.

Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking.

Communication between dying cells and their environment is a critical process that promotes tissue homeostasis during normal cellular turnover, whilst during disease settings, it can contribute to inflammation through the release of intracellular factors. Extracellular vesicles (EVs) are a heterogeneous class of membrane-bound cell-derived structures that can engage in intercellular communication via the trafficking of bioactive molecules between cells and tissues. In addition to the well-described functions of EVs derived from living cells, the ability of dying cells to release EVs capable of mediating functions on target cells or tissues is also of significant interest. In particular, during inflammatory settings such as acute tissue injury, infection and autoimmunity, the EV-mediated transfer of proinflammatory cargo from dying cells is an important process that can elicit profound proinflammatory effects in recipient cells and tissues. Furthermore, the biogenesis of EVs via unique cell-death-associated pathways has also been recently described, highlighting an emerging niche in EV biology. This review outlines the mechanisms and functions of dying-cell-derived EVs and their ability to drive inflammation during various modes of cell death, whilst reflecting on the challenges and knowledge gaps in investigating this subgenre of extracellular vesicles research.

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections.

Smac mimetics can provoke lytic cell death that is neither apoptotic nor necroptotic.

Smac mimetics, or IAP antagonists, are a class of drugs currently being evaluated as anti-cancer therapeutics. These agents antagonize IAP proteins, including cIAP1/2 and XIAP, to induce cell death via apoptotic or, upon caspase-8 deficiency, necroptotic cell death pathways. Many cancer cells are unresponsive to Smac mimetic treatment as a single agent but can be sensitized to killing in the presence of the cytokine TNFα, provided either exogenously or via autocrine production. We found that high concentrations of a subset of Smac mimetics could provoke death in cells that did not produce TNFα, despite sensitization at lower concentrations by TNFα. The ability of these drugs to kill did not correlate with valency. These cells remained responsive to the lethal effects of Smac mimetics at high concentrations despite genetic or pharmacological impairments in apoptotic, necroptotic, pyroptotic, autophagic and ferroptotic cell death pathways. Analysis of dying cells revealed necrotic morphology, which was accompanied by the release of lactate dehydrogenase and cell membrane rupture without prior phosphatidylserine exposure implying cell lysis, which occurred over a several hours. Our study reveals that cells incapable of autocrine TNFα production are sensitive to some Smac mimetic compounds when used at high concentrations, and this exposure elicits a lytic cell death phenotype that occurs via a mechanism not requiring apoptotic caspases or necroptotic effectors RIPK3 or MLKL. These data reveal the possibility that non-canonical cell death pathways can be triggered by these drugs when applied at high concentrations.

Correction to: Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis.

The original version of the article unfortunately contained a typo in the fourth author name. The author name was incorrectly listed as Rochelle Tixeria. The correct name should be Rochelle Tixeira. The original article has been corrected.

Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis.

During apoptosis, dying cells undergo dynamic morphological changes that ultimately lead to their disassembly into fragments called apoptotic bodies (ApoBDs). Reorganisation of the cytoskeletal structures is key in driving various apoptotic morphologies, including the loss of cell adhesion and membrane bleb formation. However, whether cytoskeletal components are also involved in morphological changes that occur later during apoptosis, such as the recently described generation of thin apoptotic membrane protrusions called apoptopodia and subsequent ApoBD formation, is not well defined. Through monitoring the progression of apoptosis by confocal microscopy, specifically focusing on the apoptopodia formation step, we characterised the presence of F-actin and microtubules in a subset of apoptopodia generated by T cells and monocytes. Interestingly, targeting actin polymerisation and microtubule assembly pharmacologically had no major effect on apoptopodia formation. These data demonstrate apoptopodia as a novel type of membrane protrusion that could be formed in the absence of actin polymerisation and microtubule assembly.

Moving beyond size and phosphatidylserine exposure: evidence for a diversity of apoptotic cell-derived extracellular vesicles in vitro.

Apoptosis is a form of programmed cell death that occurs throughout life as part of normal development as well as pathologic processes including chronic inflammation and infection. Although the death of a cell is often considered as the only biological outcome of a cell committed to apoptosis, it is becoming increasingly clear that the dying cell can actively communicate with other cells via soluble factors as well as membrane-bound extracellular vesicles (EVs) to regulate processes including cell clearance, immunity and tissue repair. Compared to EVs generated from viable cells such as exosomes and microvesicles, apoptotic cell-derived EVs (ApoEVs) are less well defined and the basic criteria for ApoEV characterization have not been established in the field. In this study, we will examine the current understanding of ApoEVs, in particular, the ApoEV subtype called apoptotic bodies (ApoBDs). We described that a subset of ApoBDs can be larger than 5 µm and smaller than 1 µm based on flow cytometry and live time-lapse microscopy analysis, respectively. We also described that a subset of ApoBDs can expose a relatively low level of phosphatidylserine on its surface based on annexin A5 staining. Furthermore, we characterized the presence of caspase-cleaved proteins (in particular plasma membrane-associated or cytoplasmic proteins) in samples enriched in ApoBDs. Lastly, using a combination of biochemical-, live imaging- and flow cytometry-based approaches, we characterized the progressive lysis of ApoBDs. Taken together, these results extended our understanding of ApoBDs.

Luminescent iridium(iii) complexes of N-heterocyclic carbene ligands prepared using the 'click reaction'.

A series of imidazolium salt, N-heterocyclic carbene (NHC) ligand precursors, combined with 1,2,3-triazoles were synthesized using the Cu(i) catalyzed azide-alkyne cycloaddition 'click reaction'. These pro-ligands were prepared from imidazolium molecules that were functionalized with either a terminal alkyne or azide group. Methylation of the triazole unit with methyl iodide produced a series of imidazolium/triazolium NHC pro-ligands. From these pro-ligands a family of luminescent Ir(iii) complexes of the general form [Ir(ppy)2(C^N)]+ or [Ir(ppy)2(C^C)]+ (where ppy is 2-phenylpyridine and C^N represents a bidentate imidazolylidene/triazole ligand and C^C represents a bidentate imidazolylidene/triazolylidene ligand) were prepared. The electrochemical, photophysical and electrochemiluminescence properties of the complexes have been evaluated and a preliminary study of two of these compounds as luminescent probes for cell imaging studies was conducted in A549 human lung adenocarcinoma basal epithelial cells. Co-localisation studies with the commercial dye MitoTracker CMXRos were consistent with mitochondrial uptake for these compounds.

Analysis of extracellular vesicles generated from monocytes under conditions of lytic cell death.

Extracellular vesicles (EVs) are an important class of membrane-bound structures that have been widely investigated for their roles in intercellular communication in the contexts of tumor progression, vascular function, immunity and regenerative medicine. Much of the current knowledge on the functions of EVs pertains to those derived from viable cells (e.g. exosomes and microvesicles) or apoptotic cells (e.g. apoptotic bodies) whilst the generation of EVs from dying cells under non-apoptotic conditions remains poorly characterized. Herein, the release of EVs from THP-1 monocytes under conditions of primary necrosis, secondary necrosis and pyroptosis, was investigated. A comprehensive analysis of THP-1-derived EVs revealed that cells undergoing lytic forms of cell death generated a high number of EVs compared with viable or apoptotic cells in vitro. Differential centrifugation via 16,000 g and 100,000 g revealed that dying THP-1 cells release both medium and small EVs, respectively, consistent with the known characteristics of microvesicles and/or exosomes. In addition, large EVs isolated via 2000 g centrifugation were also present in all samples. These findings suggest that lytic cell death under both sterile and non-sterile inflammatory conditions induces monocytes to generate EVs, which could potentially act as mediators of cell-to-cell communication.

Endothelial cell apoptosis and the role of endothelial cell-derived extracellular vesicles in the progression of atherosclerosis.

To maintain physiological homeostasis, cell turnover occurs every day in the body via a form of programmed cell death called apoptosis. During apoptosis, cells undergo distinct morphological changes culminating in the disassembly of the dying cell into smaller fragments known as apoptotic bodies (ApoBDs). Dysregulation of apoptosis is associated with diseases including infection, cancer and atherosclerosis. Although the development of atherosclerosis is largely attributed to the accumulation of lipids and inflammatory debris in vessel walls, it is also associated with apoptosis of macrophages, smooth muscle cells (SMCs) and endothelial cells. During cellular activation and apoptosis, endothelial cells can release several types of membrane-bound extracellular vesicles (EVs) including exosomes, microvesicles (MVs)/microparticles and ApoBDs. Emerging evidence in the field suggests that these endothelial cell-derived EVs (EndoEVs) can contribute to intercellular communication during the development of atherosclerosis via the transfer of cellular contents such as protein and microRNA, which may prevent or promote disease progression depending on the context. This review provides an up-to-date overview of the known causes and consequences of endothelial cell death during atherosclerosis along with highlighting current methodological approaches to studying EndoEVs and the potential roles of EndoEVs in atherosclerosis development.

Gasdermin E Does Not Limit Apoptotic Cell Disassembly by Promoting Early Onset of Secondary Necrosis in Jurkat T Cells and THP-1 Monocytes.

During the progression of necroptosis and pyroptosis, the plasma membrane will become permeabilized through the activation of mixed lineage kinase domain like pseudokinase (MLKL) or gasdermin D (GSDMD), respectively. Recently, the progression of apoptotic cells into secondary necrotic cells following membrane lysis was shown to be regulated by gasdermin E (GSDME, or DFNA5), a process dependent on caspase 3-mediated cleavage of GSDME. Notably, GSDME was also proposed to negatively regulate the disassembly of apoptotic cells into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs) by promoting earlier onset of membrane permeabilisation. The presence of a process downstream of caspase 3 that would actively drive cell lysis and limit cell disassembly during apoptosis is somewhat surprising as this could favor the release of proinflammatory intracellular contents and hinder efficient clearance of apoptotic materials. In contrast to the latter studies, we present here that GSDME is not involved in regulating secondary necrosis in human T cells and monocytes, and also unlikely in epithelial cells. Furthermore, GSDME is evidently not a negative regulator of apoptotic cell disassembly in our cell models. Thus, the function of GSDME in regulating membrane permeabilization and cell disassembly during apoptosis may be more limited.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Structural and Functional Insight into Canarypox Virus CNP058 Mediated Regulation of Apoptosis.

Programmed cell death or apoptosis is an important component of host defense systems against viral infection. The B-cell lymphoma 2 (Bcl-2) proteins family is the main arbiter of mitochondrially mediated apoptosis, and viruses have evolved sequence and structural mimics of Bcl-2 to subvert premature host cell apoptosis in response to viral infection. The sequencing of the canarypox virus genome identified a putative pro-survival Bcl-2 protein, CNP058. However, a role in apoptosis inhibition for CNP058 has not been identified to date. Here, we report that CNP058 is able to bind several host cell pro-death Bcl-2 proteins, including Bak and Bax, as well as several BH3 only-proteins including Bim, Bid, Bmf, Noxa, Puma, and Hrk with high to moderate affinities. We then defined the structural basis for CNP058 binding to pro-death Bcl-2 proteins by determining the crystal structure of CNP058 bound to Bim BH3. CNP058 adopts the conserved Bcl-2 like fold observed in cellular pro-survival Bcl-2 proteins, and utilizes the canonical ligand binding groove to bind Bim BH3. We then demonstrate that CNP058 is a potent inhibitor of ultraviolet (UV) induced apoptosis in a cell culture model. Our findings suggest that CNP058 is a potent inhibitor of apoptosis that is able to bind to BH3 domain peptides from a broad range of pro-death Bcl-2 proteins, and may play a key role in countering premature host apoptosis.