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1.
FASEB J ; 36(3): e22234, 2022 03.
Article in English | MEDLINE | ID: covidwho-1702985

ABSTRACT

The transmembrane protease angiotensin converting enzyme 2 (ACE2) is a protective regulator within the renin angiotensin system and additionally represents the cellular receptor for SARS-CoV. The release of soluble ACE2 (sACE2) from the cell surface is hence believed to be a crucial part of its (patho)physiological functions, as both, ACE2 protease activity and SARS-CoV binding ability, are transferred from the cell membrane to body fluids. Yet, the molecular sources of sACE2 are still not completely investigated. In this study, we show different sources and prerequisites for the release of sACE2 from the cell membrane. By using inhibitors as well as CRISPR/Cas9-derived cells, we demonstrated that, in addition to the metalloprotease ADAM17, also ADAM10 is an important novel shedding protease of ACE2. Moreover, we observed that ACE2 can also be released in extracellular vesicles. The degree of either ADAM10- or ADAM17-mediated ACE2 shedding is dependent on stimulatory conditions and on the expression level of the pro-inflammatory ADAM17 regulator iRhom2. Finally, by using structural analysis and in vitro verification, we determined for the first time that the susceptibility to ADAM10- and ADAM17-mediated shedding is mediated by the collectrin-like part of ACE2. Overall, our findings give novel insights into sACE2 release by several independent molecular mechanisms.


Subject(s)
ADAM10 Protein/metabolism , ADAM17 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Extracellular Vesicles/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , SARS Virus/metabolism , ADAM10 Protein/genetics , ADAM17 Protein/genetics , Amyloid Precursor Protein Secretases/genetics , Angiotensin-Converting Enzyme 2/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Extracellular Vesicles/genetics , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , SARS Virus/genetics , SARS-CoV-2
2.
Nat Commun ; 13(1): 405, 2022 01 20.
Article in English | MEDLINE | ID: covidwho-1631967

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of the coronavirus induced disease 2019 (COVID-19) with evolving variants of concern. It remains urgent to identify novel approaches against broad strains of SARS-CoV-2, which infect host cells via the entry receptor angiotensin-converting enzyme 2 (ACE2). Herein, we report an increase in circulating extracellular vesicles (EVs) that express ACE2 (evACE2) in plasma of COVID-19 patients, which levels are associated with severe pathogenesis. Importantly, evACE2 isolated from human plasma or cells neutralizes SARS-CoV-2 infection by competing with cellular ACE2. Compared to vesicle-free recombinant human ACE2 (rhACE2), evACE2 shows a 135-fold higher potency in blocking the binding of the viral spike protein RBD, and a 60- to 80-fold higher efficacy in preventing infections by both pseudotyped and authentic SARS-CoV-2. Consistently, evACE2 protects the hACE2 transgenic mice from SARS-CoV-2-induced lung injury and mortality. Furthermore, evACE2 inhibits the infection of SARS-CoV-2 variants (α, ß, and δ) with equal or higher potency than for the wildtype strain, supporting a broad-spectrum antiviral mechanism of evACE2 for therapeutic development to block the infection of existing and future coronaviruses that use the ACE2 receptor.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , COVID-19/immunology , Extracellular Vesicles/immunology , SARS-CoV-2/immunology , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/blood , COVID-19/epidemiology , Chlorocebus aethiops , Extracellular Vesicles/genetics , Extracellular Vesicles/metabolism , HEK293 Cells , HeLa Cells , Humans , Mice, Transgenic , Neutralization Tests/methods , Pandemics/prevention & control , Protein Binding , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Survival Analysis , Vero Cells
3.
Int J Mol Sci ; 21(19)2020 Oct 05.
Article in English | MEDLINE | ID: covidwho-1299431

ABSTRACT

CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.


Subject(s)
Extracellular Vesicles/genetics , Gene Editing/trends , Gene Transfer Techniques , RNA/genetics , CRISPR-Cas Systems , Humans , Mutation/genetics
4.
Cells ; 10(3)2021 03 04.
Article in English | MEDLINE | ID: covidwho-1125522

ABSTRACT

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.


Subject(s)
COVID-19/blood , Containment of Biohazards/methods , Extracellular Vesicles/chemistry , Virus Inactivation , COVID-19/virology , Detergents/pharmacology , Extracellular Vesicles/drug effects , Extracellular Vesicles/genetics , Hot Temperature , Humans , MicroRNAs/analysis , Microarray Analysis , Microscopy, Atomic Force , SARS-CoV-2 , Tetraspanins/analysis , Ultracentrifugation
5.
J Hematol Oncol ; 14(1): 24, 2021 02 12.
Article in English | MEDLINE | ID: covidwho-1084770

ABSTRACT

Mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have been intensely investigated for clinical applications within the last decades. However, the majority of registered clinical trials applying MSC therapy for diverse human diseases have fallen short of expectations, despite the encouraging pre-clinical outcomes in varied animal disease models. This can be attributable to inconsistent criteria for MSCs identity across studies and their inherited heterogeneity. Nowadays, with the emergence of advanced biological techniques and substantial improvements in bio-engineered materials, strategies have been developed to overcome clinical challenges in MSC application. Here in this review, we will discuss the major challenges of MSC therapies in clinical application, the factors impacting the diversity of MSCs, the potential approaches that modify MSC products with the highest therapeutic potential, and finally the usage of MSCs for COVID-19 pandemic disease.


Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Animals , Artificial Intelligence , COVID-19/therapy , CRISPR-Cas Systems , Cell Differentiation , Cell Movement , Clinical Trials as Topic , Extracellular Vesicles/genetics , Extracellular Vesicles/immunology , Extracellular Vesicles/transplantation , Genetic Engineering/methods , Humans , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/immunology , Mesenchymal Stem Cells/metabolism
6.
Int J Mol Sci ; 22(2)2021 Jan 07.
Article in English | MEDLINE | ID: covidwho-1027279

ABSTRACT

Depression is associated with an increased risk of aging-related diseases. It is also seemingly a common psychological reaction to pandemic outbreaks with forced quarantines and lockdowns. Thus, depression represents, now more than ever, a major global health burden with therapeutic management challenges. Clinical data highlights that physical exercise is gaining momentum as a non-pharmacological intervention in depressive disorders. Although it may contribute to the reduction of systemic inflammation associated with depression, the mechanisms underlying the beneficial physical exercise effects in emotional behavior remain to be elucidated. Current investigations indicate that a rapid release of extracellular vesicles into the circulation might be the signaling mediators of systemic adaptations to physical exercise. These biological entities are now well-established intercellular communicators, playing a major role in relevant physiological and pathophysiological functions, including brain cell-cell communication. We also reviewed emerging evidence correlating depression with modified circulating extracellular vesicle surfaces and cargo signatures (e.g., microRNAs and proteins), envisioned as potential biomarkers for diagnosis, efficient disease stratification and appropriate therapeutic management. Accordingly, the clinical data summarized in the present review prompted us to hypothesize that physical exercise-related circulating extracellular vesicles contribute to its antidepressant effects, particularly through the modulation of inflammation. This review sheds light on the triad "physical exercise-extracellular vesicles-depression" and suggests new avenues in this novel emerging field.


Subject(s)
Biomarkers/blood , Depression/therapy , Exercise/physiology , MicroRNAs/blood , Adaptation, Physiological/genetics , Brain/metabolism , Brain/physiology , Cell Communication/genetics , Depression/blood , Disease Management , Extracellular Vesicles/genetics , Humans
7.
Infect Genet Evol ; 85: 104422, 2020 11.
Article in English | MEDLINE | ID: covidwho-597100

ABSTRACT

Extracellular vesicles releasing from various types of cells contribute to intercellular communication via delivering bio-molecules like nucleic acids, proteins, and lipids to recipient cells. Exosomes are 30-120 nm extracellular vesicles that participate in several pathological conditions. Virus-infected cells release exosomes that are implicated in infection through transferring viral components such as viral-derived miRNAs and proteins. As well, exosomes contain receptors for viruses that make recipient cells susceptible to virus entry. Since December 2019, SARS-CoV-2 (COVID-19) infection has become a worldwide urgent public health concern. There is currently no vaccine or specific antiviral treatment existing for COVID-19 virus infection. Hence, it is critical to find a safe and effective therapeutic tool to patients with severe COVID-19 virus infection. Extracellular vesicles may contribute to spread this virus as they transfer such receptors as CD9 and ACE2, which make recipient cells susceptible to virus docking. Upon entry, COVID-19 virus may be directed into the exosomal pathway, and its component is packaged into exosomes for secretion. Exosome-based strategies for the treatment of COVID-19 virus infection may include following items: inhibition of exosome biogenesis and uptake, exosome-therapy, exosome-based drug delivery system, and exosome-based vaccine. Mesenchymal stem cells can suppress nonproductive inflammation and improve/repair lung cells including endothelial and alveolar cells, which damaged by COVID-19 virus infection. Understanding molecular mechanisms behind extracellular vesicles related COVID-19 virus infection may provide us with an avenue to identify its entry, replication, spreading, and infection to overcome its adverse effects.


Subject(s)
COVID-19/virology , Extracellular Vesicles/genetics , Extracellular Vesicles/metabolism , SARS-CoV-2/pathogenicity , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Extracellular Vesicles/drug effects , Humans , Molecular Targeted Therapy , SARS-CoV-2/drug effects , Signal Transduction/drug effects , Virus Internalization/drug effects , Virus Shedding/drug effects
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