Animal Models of Human Disease.

The use of animal models of human disease is critical for furthering our understanding of disease mechanisms, for the discovery of novel targets for treatment, and for translational research. This Special Topic entitled "Animal Models of Human Disease" aimed to collect state-of-the-art primary research studies and review articles from international experts and leading groups using animal models to study human diseases. Submissions were welcomed on a wide range of animal models and pathologies, including infectious disease, acute injury, regeneration, cancer, autoimmunity, degenerative and chronic disease. Seven participating MDPI journals supported the Special Topic, namely: Biomedicines, Cells, Current Issues in Molecular Biology, Diagnostics, Genes, the International Journal of Molecular Sciences, and the International Journal of Translational Medicine. In total, 46 papers were published in this Special Topic, with 37 full length original research papers, 2 research communications and 7 reviews. These contributions cover a wide range of clinically relevant, translatable, and comparative animal models, as well as furthering understanding of fundamental sciences, covering topics on physiological processes, on degenerative, inflammatory, infectious, autoimmune, neurological, metabolic, heamatological, hormonal and mitochondrial disorders, developmental processes and diseases, cardiology, cancer, trauma, stress, and ageing.

A Pilot Study on Peptidylarginine Deiminases and Protein Deimination in Animal Cancers across Vertebrate Species.

PADs are a group of calcium-dependent enzymes that play key roles in inflammatory pathologies and have diverse roles in cancers. PADs cause irreversible post-translational modification of arginine to citrulline, leading to changes in protein function in different cellular compartments. PAD isozyme diversity differs throughout phylogeny in chordates, with five PAD isozymes in mammals, three in birds, and one in fish. While the roles for PADs in various human cancers are mounting (both in regards to cancer progression and epigenetic regulation), investigations into animal cancers are scarce. The current pilot-study therefore aimed at assessing PAD isozymes in a range of animal cancers across the phylogeny tree. In addition, the tissue samples were assessed for total protein deimination and histone H3 deimination (CitH3), which is strongly associated with human cancers and also indicative of gene regulatory changes and neutrophil extracellular trap formation (NETosis). Cancers were selected from a range of vertebrate species: horse, cow, reindeer, sheep, pig, dog, cat, rabbit, mink, hamster, parrot, and duck. The cancers chosen included lymphoma, kidney, lung, testicular, neuroendocrine, anaplastic, papilloma, and granulosa cell tumour. Immunohistochemical analysis revealed that CitH3 was strongly detected in all of the cancers assessed, while pan-deimination detection was overall low. Both PAD2 and PAD3 were the most predominantly expressed PADs across all of the cancers assessed, while PAD1, PAD4, and PAD6 were overall expressed at lower, albeit varying, levels. The findings from this pilot study provide novel insights into PAD-mediated roles in different cancers across a range of vertebrate species and may aid in the understanding of cancer heterogeneity and cancer evolution.

Prostate cancer and microfluids.

Microfluidic systems aim to detect sample matter quickly with high sensitivity and resolution, on a small scale. With its increased use in medicine, the field is showing significant promise in prostate cancer diagnosis and management due, in part, to its ability to offer point-of-care testing. This review highlights some of the research that has been undertaken in respect of prostate cancer and microfluidics. Firstly, this review considers the diagnosis of prostate cancer through use of microfluidic systems and analyses the detection of prostate specific antigen, proteins, and circulating tumor cells to highlight the scope of current advancements. Secondly, this review analyses progressions in the understanding of prostate cancer physiology and considers techniques used to aid treatment of prostate cancer, such as the creation of a micro-environment. Finally, this review highlights potential future roles of microfluidics in assisting prostate cancer, such as in exosomal analysis. In conclusion, this review shows the vast scope and application of microfluidic systems and how these systems will ensure advancements to future prostate cancer management.

Decoy ACE2-expressing extracellular vesicles that competitively bind SARS-CoV-2 as a possible COVID-19 therapy.

The novel strain of coronavirus that appeared in 2019, SARS-CoV-2, is the causative agent of severe respiratory disease, COVID-19, and the ongoing pandemic. As for SARS-CoV that caused the SARS 2003 epidemic, the receptor on host cells that promotes uptake, through attachment of the spike (S) protein of the virus, is angiotensin-converting enzyme 2 (ACE2). In a recent article published by Batlle et al. (Clin. Sci. (Lond.) (2020) 134, 543-545) it was suggested that soluble recombinant ACE2 could be used as a novel biological therapeutic to intercept the virus, limiting the progression of infection and reducing lung injury. Another way, discussed here, to capture SARS-CoV-2, as an adjunct or alternative, would be to use ACE2+-small extracellular vesicles (sEVs). A competitive inhibition therapy could therefore be developed, using sEVs from engineered mesenchymal stromal/stem cells (MSCs), overexpressing ACE2.

Peptidylarginine Deiminase Isozyme-Specific PAD2, PAD3 and PAD4 Inhibitors Differentially Modulate Extracellular Vesicle Signatures and Cell Invasion in Two Glioblastoma Multiforme Cell Lines.

Glioblastoma multiforme (GBM) is an aggressive adult brain tumour with poor prognosis. Roles for peptidylarginine deiminases (PADs) in GBM have recently been highlighted. Here, two GBM cell lines were treated with PAD2, PAD3 and PAD4 isozyme-specific inhibitors. Effects were assessed on extracellular vesicle (EV) signatures, including EV-microRNA cargo (miR21, miR126 and miR210), and on changes in cellular protein expression relevant for mitochondrial housekeeping (prohibitin (PHB)) and cancer progression (stromal interaction molecule 1 (STIM-1) and moesin), as well as assessing cell invasion. Overall, GBM cell-line specific differences for the three PAD isozyme-specific inhibitors were observed on modulation of EV-signatures, PHB, STIM-1 and moesin protein levels, as well as on cell invasion. The PAD3 inhibitor was most effective in modulating EVs to anti-oncogenic signatures (reduced miR21 and miR210, and elevated miR126), to reduce cell invasion and to modulate protein expression of pro-GBM proteins in LN229 cells, while the PAD2 and PAD4 inhibitors were more effective in LN18 cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for deiminated proteins relating to cancer, metabolism and inflammation differed between the two GBM cell lines. Our findings highlight roles for the different PAD isozymes in the heterogeneity of GBM tumours and the potential for tailored PAD-isozyme specific treatment.

Cannabidiol Is a Novel Modulator of Bacterial Membrane Vesicles.

Membrane vesicles (MVs) released from bacteria participate in cell communication and host-pathogen interactions. Roles for MVs in antibiotic resistance are gaining increased attention and in this study we investigated if known anti-bacterial effects of cannabidiol (CBD), a phytocannabinoid from Cannabis sativa, could be in part attributed to effects on bacterial MV profile and MV release. We found that CBD is a strong inhibitor of MV release from Gram-negative bacteria (E. coli VCS257), while inhibitory effect on MV release from Gram-positive bacteria (S. aureus subsp. aureus Rosenbach) was negligible. When used in combination with selected antibiotics, CBD significantly increased the bactericidal action of several antibiotics in the Gram-negative bacteria. In addition, CBD increased antibiotic effects of kanamycin in the Gram-positive bacteria, without affecting MV release. CBD furthermore changed protein profiles of MVs released from E. coli after 1 h CBD treatment. Our findings indicate that CBD may pose as a putative adjuvant agent for tailored co-application with selected antibiotics, depending on bacterial species, to increase antibiotic activity, including via MV inhibition, and help reduce antibiotic resistance.

Peptidylarginine Deiminase Inhibitors Reduce Bacterial Membrane Vesicle Release and Sensitize Bacteria to Antibiotic Treatment.

Outer membrane and membrane vesicles (OMV/MV) are released from bacteria and participate in cell communication, biofilm formation and host-pathogen interactions. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes that catalyze post-translational deimination/citrullination of proteins, causing structural and functional changes in target proteins. PADs also play major roles in the regulation of eukaryotic extracellular vesicle release. Here we show phylogenetically conserved pathways of PAD-mediated OMV/MV release in bacteria and describe deiminated/citrullinated proteins in E. coli and their derived OMV/MVs. Furthermore, we show that PAD inhibitors can be used to effectively reduce OMV/MV release, both in Gram-negative and Gram-positive bacteria. Importantly, this resulted in enhanced antibiotic sensitivity of both E. coli and S. aureus to a range of antibiotics tested. Our findings reveal novel strategies for applying pharmacological OMV/MV-inhibition to reduce antibiotic resistance.

Mesenchymal Stromal Cell Derived Extracellular Vesicles Reduce Hypoxia-Ischaemia Induced Perinatal Brain Injury.

BACKGROUND: Neonatal hypoxic-ischemic (HI) insult is a leading cause of disability and death in newborns, with therapeutic hypothermia being the only currently available clinical intervention. Thus there is a great need for adjunct and novel treatments for enhanced or alternative post-HI neuroprotection. Extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have recently been shown to exhibit regenerative effects in various injury models. Here we present findings showing neuroprotective effects of MSC-derived EVs in the Rice-Vannucci model of severe HI-induced neonatal brain insult. METHODS: Mesenchymal stromal/stem cell-derived EVs were applied intranasally immediately post HI-insult and behavioral outcomes were observed 48 h following MSC-EV treatment, as assessed by negative geotaxis. Brains were thereafter excised and assessed for changes in glial responses, cell death, and neuronal loss as markers of damage at 48 h post HI-insult. RESULTS: Brains of the MSC-EV treated group showed a significant decrease in microglial activation, cell death, and percentage tissue volume loss in multiple brain regions, compared to the control-treated groups. Furthermore, negative geotaxis test showed improved behavioral outcomes at 48 h following MSC-EV treatment. CONCLUSION: Our findings highlight the clinical potential of using MSC-derived EVs following neonatal hypoxia-ischaemia.

Cannabidiol Affects Extracellular Vesicle Release, miR21 and miR126, and Reduces Prohibitin Protein in Glioblastoma Multiforme Cells.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary malignant brain tumor in adults, with poor prognosis. Extracellular vesicles (EVs) are key-mediators for cellular communication through transfer of proteins and genetic material. Cancers, such as GBM, use EV release for drug-efflux, pro-oncogenic signaling, invasion and immunosuppression; thus the modulation of EV release and cargo is of considerable clinical relevance. As EV-inhibitors have been shown to increase sensitivity of cancer cells to chemotherapy, and we recently showed that cannabidiol (CBD) is such an EV-modulator, we investigated whether CBD affects EV profile in GBM cells in the presence and absence of temozolomide (TMZ). Compared to controls, CBD-treated cells released EVs containing lower levels of pro-oncogenic miR21 and increased levels of anti-oncogenic miR126; these effects were greater than with TMZ alone. In addition, prohibitin (PHB), a multifunctional protein with mitochondrial protective properties and chemoresistant functions, was reduced in GBM cells following 1 h CBD treatment. This data suggests that CBD may, via modulation of EVs and PHB, act as an adjunct to enhance treatment efficacy in GBM, supporting evidence for efficacy of cannabinoids in GBM.

Cannabidiol (CBD) Is a Novel Inhibitor for Exosome and Microvesicle (EMV) Release in Cancer.

Exosomes and microvesicles (EMV) are lipid bilayer-enclosed structures, released by cells and involved in intercellular communication through transfer of proteins and genetic material. EMV release is also associated with various pathologies, including cancer, where increased EMV release is amongst other associated with chemo-resistance and active transfer of pro-oncogenic factors. Recent studies show that EMV-inhibiting agents can sensitize cancer cells to chemotherapeutic agents and reduce cancer growth in vivo. Cannabidiol (CBD), a phytocannabinoid derived from Cannabis sativa, has anti-inflammatory and anti-oxidant properties, and displays anti-proliferative activity. Here we report a novel role for CBD as a potent inhibitor of EMV release from three cancer cell lines: prostate cancer (PC3), hepatocellular carcinoma (HEPG2) and breast adenocarcinoma (MDA-MB-231). CBD significantly reduced exosome release in all three cancer cell lines, and also significantly, albeit more variably, inhibited microvesicle release. The EMV modulating effects of CBD were found to be dose dependent (1 and 5 µM) and cancer cell type specific. Moreover, we provide evidence that this may be associated with changes in mitochondrial function, including modulation of STAT3 and prohibitin expression, and that CBD can be used to sensitize cancer cells to chemotherapy. We suggest that the known anti-cancer effects of CBD may partly be due to the regulatory effects on EMV biogenesis, and thus CBD poses as a novel and safe modulator of EMV-mediated pathological events.

Peptidylarginine Deiminases Post-Translationally Deiminate Prohibitin and Modulate Extracellular Vesicle Release and MicroRNAs in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most aggressive form of adult primary malignant brain tumour with poor prognosis. Extracellular vesicles (EVs) are a key-mediator through which GBM cells promote a pro-oncogenic microenvironment. Peptidylarginine deiminases (PADs), which catalyze the post-translational protein deimination of target proteins, are implicated in cancer, including via EV modulation. Pan-PAD inhibitor Cl-amidine affected EV release from GBM cells, and EV related microRNA cargo, with reduced pro-oncogenic microRNA21 and increased anti-oncogenic microRNA126, also in combinatory treatment with the chemotherapeutic agent temozolomide (TMZ). The GBM cell lines under study, LN18 and LN229, differed in PAD2, PAD3 and PAD4 isozyme expression. Various cytoskeletal, nuclear and mitochondrial proteins were identified to be deiminated in GBM, including prohibitin (PHB), a key protein in mitochondrial integrity and also involved in chemo-resistance. Post-translational deimination of PHB, and PHB protein levels, were reduced after 1 h treatment with pan-PAD inhibitor Cl-amidine in GBM cells. Histone H3 deimination was also reduced following Cl-amidine treatment. Multifaceted roles for PADs on EV-mediated pathways, as well as deimination of mitochondrial, nuclear and invadopodia related proteins, highlight PADs as novel targets for modulating GBM tumour communication.

Peptidylarginine Deiminases-Roles in Cancer and Neurodegeneration and Possible Avenues for Therapeutic Intervention via Modulation of Exosome and Microvesicle (EMV) Release?

Exosomes and microvesicles (EMVs) are lipid bilayer-enclosed structures released from cells and participate in cell-to-cell communication via transport of biological molecules. EMVs play important roles in various pathologies, including cancer and neurodegeneration. The regulation of EMV biogenesis is thus of great importance and novel ways for manipulating their release from cells have recently been highlighted. One of the pathways involved in EMV shedding is driven by peptidylarginine deiminase (PAD) mediated post-translational protein deimination, which is calcium-dependent and affects cytoskeletal rearrangement amongst other things. Increased PAD expression is observed in various cancers and neurodegeneration and may contribute to increased EMV shedding and disease progression. Here, we review the roles of PADs and EMVs in cancer and neurodegeneration.

Chloramidine/Bisindolylmaleimide-I-Mediated Inhibition of Exosome and Microvesicle Release and Enhanced Efficacy of Cancer Chemotherapy.

Microvesicle (MV) release from tumour cells influences drug retention, contributing to cancer drug resistance. Strategically regulating MV release may increase drug retention within cancer cells and allow for lower doses of chemotherapeutic drugs. The contribution of exosomes to drug retention still remains unknown. Potential exosome and MV (EMV) biogenesis inhibitors, tested on human prostate cancer (PC3) cells for their capacity to inhibit EMV release, were also tested on PC3 and MCF-7 (breast cancer) cells for improving chemotherapy. Agents inhibiting EMV release most significantly, whilst maintaining cell viability, were chloramidine (Cl-amidine; 50 µM) and bisindolylmaleimide-I (10 µM). Apoptosis mediated by the chemotherapy drug 5-fluorouracil (5-FU) was significantly enhanced in PC3 cells in the presence of both these EMV inhibitors, resulting in a 62% (Cl-amidine + 5-FU) and 59% (bisindolylmaleimide-I + 5-FU) decrease in numbers of viable PC3 cells compared to 5-FU alone after 24 h. For MCF-7 cells, there were similar increased reductions of viable cells compared to 5-FU treatment alone ranging from 67% (Cl-amidine + 5-FU) to 58% (bisindolylmaleimide-I + 5-FU). Using combinatory treatment, the two EMV inhibitors further reduced the number of viable cancer cells tested. Neither inhibitor affected cell viability. Combining selected EMV inhibitors may pose as a novel strategy to enhance the efficacy of chemotherapeutic drug-mediated apoptosis.

The emerging role of exosome and microvesicle- (EMV-) based cancer therapeutics and immunotherapy.

There is an urgent need to develop new combination therapies beyond existing surgery, radio- and chemo-therapy, perhaps initially combining chemotherapy with the targeting specificities of immunotherapy. For this, strategies to limit inflammation and immunosuppression and evasion in the tumour microenvironment are also needed. To devise effective new immunotherapies we must first understand tumour immunology, including the roles of T cells, macrophages, myeloid suppressor cells and of exosomes and microvesicles (EMVs) in promoting angiogenesis, tumour growth, drug resistance and metastasis. One promising cancer immunotherapy discussed uses cationic liposomes carrying tumour RNA (RNA-lipoplexes) to provoke a strong anti-viral-like (cytotoxic CD8+ ) anti-tumour immune response. Mesenchymal stem cell-derived EMVs, with their capacity to migrate towards inflammatory areas including solid tumours, have also been used. As tumour EMVs clearly exacerbate the tumour microenvironment, another therapy option could involve EMV removal. Affinity-based methods to deplete EMVs, including an immunodepletion, antibody-based affinity substrate, are therefore considered. Finally EMV and exosome-mimetic nanovesicles (NVs) delivery of siRNA or chemotherapeutic drugs that target tumours using peptide ligands for cognate receptors on the tumour cells are discussed. We also touch upon the reversal of drug efflux in EMVs from cancer cells which can sensitize cells to chemotherapy. The use of immunotherapy in combination with the advent of EMVs provides potent therapies to various cancers.

A novel role for peptidylarginine deiminases in microvesicle release reveals therapeutic potential of PAD inhibition in sensitizing prostate cancer cells to chemotherapy.

INTRODUCTION: Protein deimination, defined as the post-translational conversion of protein-bound arginine to citrulline, is carried out by a family of 5 calcium-dependent enzymes, the peptidylarginine deiminases (PADs) and has been linked to various cancers. Cellular microvesicle (MV) release, which is involved in cancer progression, and deimination have not been associated before. We hypothesize that elevated PAD expression, observed in cancers, causes increased MV release in cancer cells and contributes to cancer progression. BACKGROUND: We have previously reported that inhibition of MV release sensitizes cancer cells to chemotherapeutic drugs. PAD2 and PAD4, the isozymes expressed in patients with malignant tumours, can be inhibited with the pan-PAD-inhibitor chloramidine (Cl-am). We sought to investigate whether Cl-am can inhibit MV release and whether this pathway could be utilized to further increase the sensitivity of cancer cells to drug-directed treatment. METHODS: Prostate cancer cells (PC3) were induced to release high levels of MVs upon BzATP stimulation of P2X7 receptors. Western blotting with the pan-protein deimination antibody F95 was used to detect a range of deiminated proteins in cells stimulated to microvesiculate. Changes in deiminated proteins during microvesiculation were revealed by immunoprecipitation and immunoblotting, and mass spectrometry identified deiminated target proteins with putative roles in microvesiculation. CONCLUSION: We report for the first time a novel function of PADs in the biogenesis of MVs in cancer cells. Our results reveal that during the stimulation of prostate cancer cells (PC3) to microvesiculate, PAD2 and PAD4 expression levels and the deimination of cytoskeletal actin are increased. Pharmacological inhibition of PAD enzyme activity using Cl-am significantly reduced MV release and abrogated the deimination of cytoskeletal actin. We demonstrated that combined Cl-am and methotrexate (MTX) treatment of prostate cancer cells increased the cytotoxic effect of MTX synergistically. Refined PAD inhibitors may form part of a novel combination therapy in cancer treatment.

Exosomes serve as tumour markers for personalized diagnostics owing to their important role in cancer metastasis.

Exosomes, membrane vesicles of 40-100 nm in diameter, are derived from endosomes in various cells. The bioactive molecules specifically packed into exosomes can be horizontally transferred into recipient cells changing their biological properties, by which tumour cells continuously modify their surrounding microenvironment and distant target cells favouring cancer metastasis. It has been suspected for a long time that exosomes participate in the whole process of tumour metastasis. Although there is much unknown and many controversies in the role of cancer exosome, the major contribution of tumour-associated exosomes to different steps of cancer metastasis are demonstrated in this review. Mainly because these exosomes are easily accessible and capable of representing their parental cells, exosomes draw much attention as a promising biomarker for tumour screening, diagnosis and prognosis. Currently, researchers have found numerous biomarkers in exosomes with great potential to be utilized in personalized medicine. In this article, we summarize the roles of biomarkers, which are validated by clinical samples. Even though many conundrums remain, such as exosome extraction, large multicentre validation of biomarkers and data interpretation, exosomes are certain to be used in clinical practice in the near future as the field rapidly expands.

Blood/plasma secretome and microvesicles.

A major but hitherto overseen component of the blood/plasma secretome is that of extracellular vesicles (EVs) which are shed from all blood cell types. These EVs are made up of microvesicles (MVs) and exosomes. MVs, 100nm-1µm in diameter, are released from the cell surface, and are a rich source of non-conventionally secreted proteins lacking a conventional signal peptide, and thus not secreted by the classical secretory pathways. Exosomes are smaller vesicles (≤100nm) having an endocytic origin and released upon multivesicular body fusion with the plasma membrane. Both vesicle types play major roles in intercellular cross talk and constitute an important component of the secretome especially in the area of biomarkers for cancer. The release of EVs, which are found in all the bodily fluids, is enhanced in cancer and a major focus of cancer proteomics is therefore targeted at EVs. The blood/plasma secretome is also a source of EVs, potentially diagnostic of infectious disease, whether from EVs released from infected cells or from the pathogens themselves. Despite the great excitement in this field, as is stated here and in other parts of this Special issue entitled: An Updated Secretome, much of the EV research, whether proteomic or functional in nature, urgently needs standardisation both in terms of nomenclature and isolation protocols. This article is part of a Special Issue entitled: An Updated Secretome.

Microvesiculation and disease.

The important roles of extracellular vesicles in the pathogenesis of various diseases are rapidly being elucidated. As important vehicles of intercellular communication, extracellular vesicles, which comprise microvesicles and exosomes, are revealing important roles in cancer tumorigenesis and metastases and in the spread of infectious disease. The September 2012 Focused Meeting 'Microvesiculation and Disease' brought together researchers working on extracellular vesicles. The papers in this issue of Biochemical Society Transactions review work in areas including HIV infection, kidney disease, hypoxia-mediated tumorigenesis and down-regulation of immune cell functions in acute myeloid leukaemia by tumour-derived exosomes. In all cases, microvesicles and exosomes have been demonstrated to be important factors leading to the pathophysiology of disease or indeed as therapeutic vehicles in possible new treatments. The aim was, having enhanced our molecular understanding of the contribution of microvesicles and exosomes to disease in vitro, to begin to apply this knowledge to in vivo models of disease.

Interplay of host-pathogen microvesicles and their role in infectious disease.

The release of extracellular vesicles, whether MVs (microvesicles) or exosomes, from host cells or intracellular pathogens is likely to play a significant role in the infection process. Host MVs may fuse with pathogen surfaces to deliver host complement regulatory proteins. They may also deliver cytokines that enhance invasion. Decoy functions are also possible. Whereas host MVs may direct pathogens away from their target cells, pathogen MVs may in turn redirect complement membrane-attack complexes away from their target pathogen. An understanding of the mechanisms of this interplay, bringing about both immune evasion and enhanced invasion, will help to direct future research with a view to rendering pathogens more susceptible to immune attack or in improving drug efficacy. It should also be possible to use MVs or exosomes isolated directly from the pathogens, or from the cells infected with pathogens, to provide alternative vaccination strategies.

The role of microvesicles in cancer progression and drug resistance.

Microvesicles are shed constitutively, or upon activation, from both normal and malignant cells. The process is dependent on an increase in cytosolic Ca2+, which activates different enzymes, resulting in depolymerization of the actin cytoskeleton and release of the vesicles. Drug resistance can be defined as the ability of cancer cells to survive exposure to a wide range of anti-cancer drugs, and anti-tumour chemotherapeutic treatments are often impaired by innate or acquired MDR (multidrug resistance). Microvesicles released upon chemotherapeutic agents prevent the drugs from reaching their targets and also mediate intercellular transport of MDR proteins.