Peer Review #2 of "Characterization and function of medium and large extracellular vesicles from plasma and urine by surface antigens and Annexin V (v0.3)"

AbstractMedium/large extracellular vesicles (m/lEVs) are released by most cell types and are involved in multiple basic biological processes. Analysis of m/lEV levels in blood or urine may help unravel pathophysiological findings in many diseases. However, it remains unclear how many naturally-occurring m/lEV subtypes exist as well as how their characteristics and functions differ from one another. Here, we identified m/lEVs pelleted from plasma and urine samples by differential centrifugation and showed by flow cytometry that they typically possessed diameters between 200 nm and 800 nm. Using proteomic profiling, we identified several proteins involved in m/lEV biogenesis including adhesion molecules, peptidases and exocytosis regulatory proteins. In healthy human plasma, we could distinguish m/lEVs derived from platelets, erythrocytes, monocytes/macrophages, T and B cells, and vascular endothelial cells using various surface antigens. m/lEVs derived from erythrocytes and monocytes were Annexin V positive. In urine, 50% of m/lEVs were Annexin V negative but contained various membrane peptidases derived from renal tubular villi. Urinary m/lEVs, but not plasma m/lEVs, showed peptidase activity. The method we have developed to characterize cell-derived m/lEVs suggests the possibility of clinical applications.

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Characterization and function of medium and large extracellular vesicles from plasma and urine by surface antigens and Annexin V

10.7717/peerj-achem.4 ◽

2020 ◽

Vol 2 ◽

pp. e4 ◽

Cited By ~ 3

Author(s):

Ko Igami ◽

Takeshi Uchiumi ◽

Saori Ueda ◽

Kazuyuki Kamioka ◽

Daiki Setoyama ◽

...

Keyword(s):

Extracellular Vesicles ◽

Vascular Endothelial Cells ◽

Membrane Surface ◽

Annexin V ◽

Shotgun Proteomics ◽

Surface Antigens ◽

Flow Cytometer ◽

Peptidase Activity ◽

Proteomic Profiling ◽

Healthy Human

Background Extracellular vesicles (EVs) are released by most cell types and are involved in multiple basic biological processes. Medium/large EVs (m/lEVs), which are of a different size from exosomes, play an important role in the coagulation in blood, and are secreted from cancer cells, etc., suggesting functions related to malignant transformation. The m/lEVs levels in blood or urine may help unravel pathophysiological findings in many diseases. However, it remains unclear how many naturally-occurring m/lEV subtypes exist as well as how their characteristics and functions differ from one another. Methods We used the blood and urinal sample from each 10 healthy donors for analysis. Using a flow cytometer, we focus on characterization of EVs with large sizes (>200 nm) that are different from exosomes. We also searched for a membrane protein for characterization with a flow cytometer using shotgun proteomics. We then identified m/lEVs pelleted from plasma and urine samples by differential centrifugation and characterized by flow cytometry. Results Using proteomic profiling, we identified several proteins involved in m/lEV biogenesis including adhesion molecules, peptidases and exocytosis regulatory proteins. In healthy human plasma, we could distinguish m/lEVs derived from platelets, erythrocytes, monocytes/macrophages, T and B cells, and vascular endothelial cells with more than two positive surface antigens. The ratio of phosphatidylserine appearing on the membrane surface differed depending on the cell-derived m/lEVs. In urine, 50% of m/lEVs were Annexin V negative but contained various membrane peptidases derived from renal tubular villi. Urinary m/lEVs, but not plasma m/lEVs, showed peptidase activity. The knowledge of the new characteristics is considered to be useful as a diagnostic material and the newly developed method suggests the possibility of clinical application.

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Extracellular vesicles as mediators and markers of acute organ injury: current concepts

European Journal of Trauma and Emergency Surgery ◽

10.1007/s00068-021-01607-1 ◽

2021 ◽

Author(s):

Birte Weber ◽

Niklas Franz ◽

Ingo Marzi ◽

Dirk Henrich ◽

Liudmila Leppik

Keyword(s):

Extracellular Vesicles ◽

Inflammatory Diseases ◽

Organ Injury ◽

Isolation And Characterization ◽

Communication Processes ◽

Incidence And Mortality ◽

New Biomarkers ◽

And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

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Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice

Biology ◽

10.3390/biology10010029 ◽

2021 ◽

Vol 10 (1) ◽

pp. 29

Author(s):

Raghubendra Singh Dagur ◽

Moses New-Aaron ◽

Murali Ganesan ◽

Weimin Wang ◽

Svetlana Romanova ◽

...

Keyword(s):

Oxidative Stress ◽

Extracellular Vesicles ◽

Treated Group ◽

Human Hepatocytes ◽

In Vivo Studies ◽

People Living With Hiv ◽

Humanized Mouse Model ◽

And Function

Background: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods:The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. Results: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs’ generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

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