Necrotic debris and STING exert therapeutically relevant effects on tumor cholesterol homeostasis

Malignant tumors commonly display necrosis, which invariably triggers an inflammatory response that supports tumor growth. However, the effect on tumor cells of necrotic debris, or damage-associated molecular patterns (DAMPs) released by dying cells is unknown. Here, we addressed the effect of DAMPs on primary Ewing sarcoma (EwS) cells and cell lines grown in 3D (spheroids) and 2D culture. We show that DAMPs promote the growth of EwS spheroids but not 2D cultures and that the underlying mechanism implicates an increase in cholesterol load in spheroids. In contrast, stimulation of the nucleic acid sensor signaling platform STING by its ligand cyclic GMP-AMP decreases the tumor cell cholesterol load and reduces their tumor initiating ability. Overexpression of STING or stimulation with cyclic GMP-AMP opposes the growth stimulatory effect of DAMPs and synergizes with the cholesterol synthesis inhibitor simvastatin to inhibit tumor growth. Our observations show that modulation of cholesterol homeostasis is a major effect of necrotic cell debris and STING and suggest that combining STING agonists with statins may help control tumor growth.

Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells

Several hundred billion to a trillion cells die in the body every day and release cell free chromatin particles (cfChPs) which enter into the circulation, or are released locally into extracellular compartments of the body. We have reported that cfChPs from the dying cells can readily enter into living cells and damage their DNA. To test the hypothesis that internalised cfChPs might also inflict mitochondrial damage, we treated NIH3T3 mouse fibroblast cells with cfChPs isolated from sera of healthy individuals (10ng), or co-cultured the cells with hypoxia induced dying NIH3T3 cells. Abundant cfChPs could be detected in the cytoplasm of the treated cells by 4h. The latter was associated with evidence of mitochondrial damage in the form of ultra-structural changes, increased mitochondrial mass, alterations in mitochondrial shape, upregulation of the mitochondrial outer membrane protein TOM20, and changes in mitochondrial membrane potential. We also detected increased fluorescence signals of gamma-H2AX and p-ATM signifying double-strand breaks in mitochondrial DNA. There was marked increase in production of mitochondrial superoxide (ROS) as detected by MitoSOX Red, and activation of the intracellular antioxidant enzyme superoxide dismutase-1. Mitochondrial damage and ROS production could be inhibited by a cfChPs deactivating agent viz. anti-histone antibody complexed nanoparticles. Given that 1x109-1x1012 cells die in the body every day, we propose that cfChPs are major physiological triggers for mitochondrial damage and ROS production with an important bearing on human health and disease. Deactivation of cfChPs may provide a novel therapeutic approach to retard ageing and associated degenerative conditions that have been linked to oxidative stress.

Supplemental Oxygen in the Newborn: Historical Perspective and Current Trends

Oxygen is the final electron acceptor in aerobic respiration, and a lack of oxygen can result in bioenergetic failure and cell death. Thus, administration of supplemental concentrations of oxygen to overcome barriers to tissue oxygen delivery (e.g., heart failure, lung disease, ischemia), can rescue dying cells where cellular oxygen content is low. However, the balance of oxygen delivery and oxygen consumption relies on tightly controlled oxygen gradients and compartmentalized redox potential. While therapeutic oxygen delivery can be life-saving, it can disrupt growth and development, impair bioenergetic function, and induce inflammation. Newborns, and premature newborns especially, have features that confer particular susceptibility to hyperoxic injury due to oxidative stress. In this review, we will describe the unique features of newborn redox physiology and antioxidant defenses, the history of therapeutic oxygen use in this population and its role in disease, and clinical trends in the use of therapeutic oxygen and mitigation of neonatal oxidative injury.

Preclinical assessment of [68Ga]Ga-Cell Death Indicator (CDI): a novel hsp90 ligand for positron emission tomography of cell death

Background: 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO) when conjugated with a bifunctional chelator 2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODAGA) (hereafter referred to as Cell Death Indicator [CDI]), enters dead and dying cells and binds to 90kDa heat shock proteins (hsp90). Objective: This study assesses stability, biodistribution, imaging, and radiation dosimetry of [68Ga]Ga-CDI for positron emission tomography (PET). Methods: Preparation of [68Ga]Ga-CDI was performed as previously described. Product stability and stability in plasma were assessed using high-performance liquid chromatography. Biodistribution and imaging were conducted in ten healthy male Lewis rats at 1 and 2 h following intravenous [68Ga]Ga-CDI injection. Human radiation dosimetry was estimated by extrapolation for a standard reference man and calculated with OLINDA/EXM 1.1. Results: Radiochemical purity of [68Ga]Ga-CDI averaged 93.8% in the product and 86.7% in plasma at 4 h post-synthesis. The highest concentration of [68Ga]Ga-CDI is observed in the kidneys; [68Ga]Ga-CDI is excreted in the urine, and mean retained activity was 32.4% and 21.4% at 1 and 2 h post-injection. Lower concentrations of [68Ga]Ga-CDI were present in the small bowel and liver. PET CT was concordant and additionally demonstrated focal growth plate uptake. The effective dose for [68Ga]Ga-CDI is 2.16E-02 mSv/MBq, and the urinary bladder wall received the highest dose (1.65E-02mSv/Mbq). Conclusions: [68Ga]Ga-CDI is stable and has favourable biodistribution, imaging, and radiation dosimetry for imaging of dead and dying cells. Human studies are underway.

Evolution of bone tissue based on angiogenesis as a crucial factor: New mathematical attempt

In this paper, we present a novel theoretical model of bone evolution. The model takes into account growth of the blood vessels network as a real issue during the process. The stimulation of this growth is controlled by the signals from overloaded dying cells. A system of integro-differential equations describes changes in the density of blood vessels, bone cells and Young modulus, all of which define how the bone tissue evolves over time. The model considers several biomechanical signals such as changes in the density of elastic strain energy and nutrients. Two examples of changing bone tissue were examined to test the model: bone healing around a tooth implant and formation of osteophytes during osteoarthritis. In both cases, the effects of mechanical loading in the bone tissue evolution which are in agreement with the medical observations can be observed.

Efferocytosis: An Interface between Apoptosis and Pathophysiology

Several cell death modes, each with a unique feature and mode of inducing cell death have been established. Cell death occurring under physiological conditions is primarily caused by apoptosis, which is a non-inflammatory or silent process, whereas necroptosis or pyroptosis is triggered by pathogen invasion, which stimulates the immune system and induces inflammation. In physiology, clearing dead cells and associated cellular debris is necessary since billions of cells die during mammalian embryogenesis and every day in adult organisms. For degradation, dead cells produced by apoptosis are quickly engulfed by macrophages. This chapter will present a description of the phagocytosis of dead and dying cells, by a process known as efferocytosis. Macrophages and, to a lesser degree, other ‘professional’ phagocytes (such as monocytes and dendritic cells) and ‘non-professional’ phagocytes, such as epithelial cells, conduct efferocytosis. Recent discoveries have shed light on this mechanism and how it works to preserve homeostasis of tissue, repair of tissue and health of the organism. Caspases are a large family of proteases of cysteine acting in cascades. A cascade leading to activation of caspase 3 mediates apoptosis and is responsible for killing cells, hiring macrophages, and presenting a “eat me” signal(s). If macrophages do not effectively engulf apoptotic cells, they undergo secondary necrosis and release intracellular materials that reflect a molecular pattern associated with injury, which can lead to autoimmune diseases. Here, the processes of efferocytosis are illustrated and the pathophysiological effects that which occur when this phase is abrogated are highlighted.

Planarians as an In Vivo Experimental Model for the Study of New Radioprotective Substances

Ionising radiation causes the death of the most actively dividing cells, thus leading to depletion of the stem cell pool. Planarians are invertebrate flatworms that are unique in that their stem cells, called neoblasts, constantly replace old, damaged, or dying cells. Amenability to efficient RNAi treatments, the rapid development of clear phenotypes, and sensitivity to ionising radiation, combined with new genomic technologies, make planarians an outstanding tool for the discovery of potential radioprotective agents. In this work, using the well-known antioxidant N-acetylcysteine, planarians are, for the first time, shown to be an excellent model system for the fast and effective screening of novel radioprotective and radio-sensitising substances. In addition, a panel of measurable parameters that can be used for the study of radioprotective effects on this model is suggested.

Role of Gasdermins in the Biogenesis of Apoptotic Cell–Derived Exosomes

The gasdermins are a family of pore-forming proteins that has recently been suggested to play a central role in the pyroptosis and the release of inflammatory cytokines. Here, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell–derived exosomes. In apoptotic cells, GADMA, GSDMC, GSDMD, and GSDME increased the release of ApoExos, and both their full-length and cleaved forms were localized in the exosomal membrane. GSDMB and DFNB59, on the other hand, negatively affected the release of ApoExos. The caspase-mediated cleavage of gasdermins, especially GSDME, is suggested to increase Ca2+ influx to cytosol through endosomal pores and thus increase the biogenesis of ApoExos. In addition, the GSDME-mediated biogenesis of ApoExos depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins: annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of ApoExos begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that GSDME-bearing tumors released ApoExos to induce inflammatory responses in the in vivo 4T1 orthotropic model of breast cancer. The data presented in this study indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4

Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, is involved in the breakage of self-tolerance in anti-CCP-positive rheumatoid arthritis. This reaction is catalyzed by peptidyl arginine deiminases (PADs), of which PAD2 and PAD4 are thought to play key pathogenic roles. Small-molecule PAD inhibitors such as the pan-PAD inhibitor BB-Cl-amidine, the PAD2-specific inhibitor AFM-30a, and the PAD4-specific inhibitor GSK199 hold therapeutic potential and are useful tools in studies of citrullination. Using an ELISA based on the citrullination of fibrinogen, we found that AFM-30a inhibited the catalytic activity of PADs derived from live PMNs or lysed PBMCs and PMNs and of PADs in cell-free synovial fluid samples from RA patients, while GSK199 had minor effects. In combination, AFM-30a and GSK199 inhibited total intracellular citrullination and citrullination of histone H3 in PBMCs, as determined by Western blotting. They were essentially nontoxic to CD4+ T cells, CD8+ T cells, B cells, NK cells, and monocytes at concentrations ranging from 1 to 20 μM, while BB-Cl-amidine was cytotoxic at concentrations above 1 μM, as assessed by flow cytometric viability staining and by measurement of lactate dehydrogenase released from dying cells. In conclusion, AFM-30a is an efficient inhibitor of PAD2 derived from PBMCs, PMNs, or synovial fluid. AFM-30a and GSK199 can be used in combination for inhibition of PAD activity associated with PBMCs but without the cytotoxic effect of BB-Cl-amidine. This suggests that AFM-30a and GSK199 may have fewer off-target effects than BB-Cl-amidine and therefore hold greater therapeutic potential.