Microvesicles as promising biological tools for diagnosis and therapy

ABSTRACT

Introduction: Shed by most cells, in response to a myriad of stimuli, extracellular vesicles (EVs) carry proteins, lipids, and various nucleic acids. EVs encompass diverse subpopulations differing for biogenesis and content. Among these, microvesicles (MVs) derived from plasma membrane, are key regulators of physiopathological cellular processes including cancer, inflammation and infection. This review is unique in that it focuses specifically on the MVs as a mediator of information transfer. In fact, few proteomic studies have rigorously distinguished MVs from exosomes.

Areas covered: Aim of this review is to discuss the proteomic analyses of the MVs. Many studies have examined mixed populations containing both exosomes and MVs. We discuss MVs’ role in cell-specific interactions. We also show their emerging roles in therapy and diagnosis.

Expert commentary: We see MVs as therapeutic tools for potential use in precision medicine. They may also have potential for allowing the identification of new biomarkers. MVs represent an invaluable tool for studying the cell of origin, which they closely represent, but it is critical to build a repository with data from MVs to deepen our understanding of their molecular repertoire and biological functions.     

Discovery of novel Bcr–Abl inhibitors targeting myristoyl pocket and ATP site

Bcr–Abl plays an essential role in the pathogenesis and development of chronic myeloid leukaemia (CML). Inhibition of Bcr–Abl has great potential for therapeutic intervention in CML. In order to obtain novel and potent Bcr–Abl inhibitors, twenty seven 4,6-disubstituted pyrimidines were synthesized and evaluated herein. The biological results indicated that four compounds of them (C4, C5, C16, and C23) were potent Bcr–Abl inhibitors which were comparable to positive control. Moreover, C4 and C5 displayed promising antiproliferative activity against K562 cells. The results suggested that these 4,6-disubstituted pyrimidines could serve as promising leads for further optimization of Bcr–Abl inhibitors.     

Glutaminase-containing microvesicles from HIV-1-infected macrophages and immune-activated microglia induce neurotoxicity

Background

HIV-1-infected and/or immune-activated microglia and macrophages are pivotal in the pathogenesis of HIV-1-associated neurocognitive disorders (HAND). Glutaminase, a metabolic enzyme that facilitates glutamate generation, is upregulated and may play a pathogenic role in HAND. Our previous studies have demonstrated that glutaminase is released to the extracellular fluid during HIV-1 infection and neuroinflammation. However, key molecular mechanisms that regulate glutaminase release remain unknown. Recent advances in understanding intercellular trafficking have identified microvesicles (MVs) as a novel means of shedding cellular contents. We posit that during HIV-1 infection and immune activation, microvesicles may mediate glutaminase release, generating excessive and neurotoxic levels of glutamate.

Results

MVs isolated through differential centrifugation from cell-free supernatants of monocyte-derived macrophages (MDM) and BV2 microglia cell lines were first confirmed in electron microscopy and immunoblotting. As expected, we found elevated number of MVs, glutaminase immunoreactivities, as well as glutaminase enzyme activity in the supernatants of HIV-1 infected MDM and lipopolysaccharide (LPS)-activated microglia when compared with controls. The elevated glutaminase was blocked by GW4869, a neutral sphingomyelinase inhibitor known to inhibit MVs release, suggesting a critical role of MVs in mediating glutaminase release. More importantly, MVs from HIV-1-infected MDM and LPS-activated microglia induced significant neuronal injury in rat cortical neuron cultures. The MV neurotoxicity was blocked by a glutaminase inhibitor or GW4869, suggesting that the neurotoxic potential of HIV-1-infected MDM and LPS-activated microglia is dependent on the glutaminase-containing MVs.

Conclusions

These findings support MVs as a potential pathway/mechanism of excessive glutamate generation and neurotoxicity in HAND and therefore MVs may serve as a novel therapeutic target.

     

Microvesicle-mediated Transfer of MicroRNA-150 from Monocytes to Endothelial Cells Promotes Angiogenesis

Recent studies by our group and others show that microRNAs can be actively secreted into the extracellular environment through microvesicles (MVs) and function as secretory signaling molecules that influence the recipient cell phenotypes. Here we investigate the role of monocyte-secreted miR-150 in promoting the capillary tube formation of endothelial cells and in enhancing angiogenesis. In vitro capillary tube formation and in vivo angiogenesis assays showed that monocyte-derived MVs have strong pro-angiogenic activities. By depleting miR-150 from monocytic MVs and increasing miR-150 in MVs derived from cells that normally contain low levels of miR-150, we further demonstrated that the miR-150 content accounted for the pro-angiogenic activity of monocytic MVs in these assays. Using tumor-implanted mice and ob/ob mice as models, we revealed that miR-150 secretion, which is increased for diseases such as cancers and diabetes, significantly promotes angiogenesis. The delivery of anti-miR-150 antisense oligonucleotides into tumor-implanted mice and ob/ob mice via MVs, however, strongly reduced angiogenesis in both types of mice. Our results collectively demonstrate that secretion of miR-150 via MVs can promote angiogenesis in vitro and in vivo, and we also present a novel microRNA-based therapeutic approach for disease treatment.     

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis,properties, functions and biomimetic models

BackgroundMatrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100–300 nm diameter harboring the biochemical machinery needed to induce mineralization.Scope of the reviewThe review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs.Major conclusionsMVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies.General significanceMVs have been extensively investigated owing to their roles in skeletal and ectopic mineralization. MVs serve as a model system for lipid raft structures, and for the mechanisms of genesis and release of extracellular vesicles.     

Colorectal cancer cell-derived microvesicles containing microRNA-1246 promote angiogenesis by activating Smad 1/5/8 signaling elicited by PML down-regulation in endothelial cells

Emerging studies on circulating microRNAs (miRNAs) or microvesicles (MVs) have shown the potential of them to be novel biomarkers and therapeutic targets for cancer. However, the biological roles of these miRNAs and MVs have not been validated yet. To determine the biological significance of MVs, we used human colorectal cancer cells as the MV donor and endothelial cells (HUVECs) as the MV recipient and demonstrated the transfer of colorectal cancer cell-derived MVs (CRC-MVs) to HUVECs and evaluated the roles of these MVs and their cargo in tumor angiogenesis. Consequently, the incubation of HUVECs with CRC-MVs promoted the proliferation, migration, and tube formation activities of these cells. Among the cargoes shuttled by the MVs, miR-1246 and TGF-β were considered to be responsible for the pro-angiogenic function of MVs by activating Smad 1/5/8 signaling in the HUVECs. These results suggest that colorectal cancer cells secreted MVs to contribute to tumor angiogenesis.     

A Novel Role for Pro-Coagulant Microvesicles in the Early Host Defense against Streptococcus pyogenes

Previous studies have shown that stimulation of whole blood or peripheral blood mononuclear cells with bacterial virulence factors results in the sequestration of pro-coagulant microvesicles (MVs). These particles explore their clotting activity via the extrinsic and intrinsic pathway of coagulation; however, their pathophysiological role in infectious diseases remains enigmatic. Here we describe that the interaction of pro-coagulant MVs with bacteria of the species Streptococcus pyogenes is part of the early immune response to the invading pathogen. As shown by negative staining electron microscopy and clotting assays, pro-coagulant MVs bind in the presence of plasma to the bacterial surface. Fibrinogen was identified as a linker that, through binding to the M1 protein of S. pyogenes, allows the opsonization of the bacteria by MVs. Surface plasmon resonance analysis revealed a strong interaction between pro-coagulant MVs and fibrinogen with a K_D value in the nanomolar range. When performing a mass-spectrometry-based strategy to determine the protein quantity, a significant up-regulation of the fibrinogen-binding integrins CD18 and CD11b on pro-coagulant MVs was recorded. Finally we show that plasma clots induced by pro-coagulant MVs are able to prevent bacterial dissemination and possess antimicrobial activity. These findings were confirmed by in vivo experiments, as local treatment with pro-coagulant MVs dampens bacterial spreading to other organs and improved survival in an invasive streptococcal mouse model of infection. Taken together, our data implicate that pro-coagulant MVs play an important role in the early response of the innate immune system in infectious diseases.     

Multifunctional membrane vesicles in Pseudomonas aeruginosa

Gram‐negative bacteria secrete small particles called membrane vesicles (MVs) into the extracellular milieu. While MVs have important roles in delivering toxins from pathogenic bacteria to eukaryotic cells, these vesicles also play ecological roles necessary for survival in various environmental conditions. Pseudomonas aeruginosa, which lives in soil, ocean, plant, animal and human environments, has become a model organism for studying these small extracellular particles. Such studies have increased our understanding of the function and biogenesis of bacterial MVs. Pseudomonas aeruginosa MVs possess versatile components and chemical substances with unique structures. These characteristics allow MVs to play their multifunctional biological roles, including microbial interaction, maintenance of biofilm structure and host infection. This review summarizes the comprehensive biochemical and physiochemical properties of MVs derived from P. aeruginosa. These studies will help us understand their biological roles of MVs not only in pathogenicity but also in microbial ecology. Also, the mechanisms of MV production, as currently understood, are discussed.     

Membrane vesicles from a Dietzia bacterium containing multiple cargoes and their roles in iron delivery

Membrane vesicles (MVs) released from bacteria act as extracellular vehicles carrying various functional cargoes between cells. MVs with different cargoes play multiple roles in stress adaptation, nutrient acquisition and microbial interactions. However, previous studies have primarily focused on MVs from Gram-negative bacteria, while the characteristics of cargoes in MVs from Gram-positive bacteria and their involvement in microbial interactions remain to be elucidated. Here, we used a Gram-positive strain, Dietzia sp. DQ12-45-1b from Corynebacteriales, to analyse the characteristics and functions of MVs. We identified the ‘antioxidant’ canthaxanthin is stored within MVs by LC–MS/MS. In addition, nearly the entire genomic content of strain DQ12-45-1b are evenly distributed in MVs, suggesting that MVs from DQ12-45-1b might involve in horizontal gene transfer. Finally, the mycobactin-type siderophores were detected in MVs. The iron-loaded MVs effectively mediate iron binding and delivery to homologous bacteria from the order Corynebacteriales, but not to more distantly related species from the orders Pseudomonadales, Bacillales and Enterobacterales. These results revealed that the iron-loaded MVs are shared between homologous species. Together, we report the Gram-positive bacterium Dietzia sp. DQ12-45-1b released MVs that contain canthaxanthin, DNA and siderophores and prove that MVs act as public goods between closely related species.     

Elevated Levels of Procoagulant Plasma Microvesicles in Dialysis Patients

Cardiovascular (CV) death remains the largest cause of mortality in dialysis patients, unexplained by traditional risk factors. Endothelial microvesicles (EMVs) are elevated in patients with traditional CV risk factors and acute coronary syndromes while platelet MVs (PMVs) are associated with atherosclerotic disease states. This study compared relative concentrations of circulating MVs from endothelial cells and platelets in two groups of dialysis patients and matched controls and investigated their relative thromboembolic risk. MVs were isolated from the blood of 20 haemodialysis (HD), 17 peritoneal dialysis (PD) patients and 20 matched controls. Relative concentrations of EMVs (CD144^{+ ve}) and PMVs (CD42b^{+ ve}) were measured by Western blotting and total MV concentrations were measured using nanoparticle-tracking analysis. The ability to support thrombin generation was measured by reconstituting the MVs in normal plasma, using the Continuous Automated Thrombogram assay triggered with 1µM tissue factor. The total concentration of MVs as well as the measured sub-types was higher in both patient groups compared to controls (p<0.05). MVs from HD and PD patients were able to generate more thrombin than the controls, with higher peak thrombin, and endogenous thrombin potential levels (p<0.02). However there were no differences in either the relative quantity or activity of MVs between the two patient groups (p>0.3). Dialysis patients have higher levels of circulating procoagulant MVs than healthy controls. This may represent a novel and potentially modifiable mediator or predictor of occlusive cardiovascular events in these patients.     

Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34⁺ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34⁺ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34⁺ cells as well as viability and clonogenic assays of CD34⁺ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34⁺ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34⁺ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.     

Omics Approaches to Identify Potential Biomarkers of Inflammatory Diseases in the Focal Adhesion Complex

Inflammatory diseases such as inflammatory bowel disease(IBD) require recurrent invasive tests, including blood tests, radiology, and endoscopic evaluation both to diagnose and assess disease activity, and to determine optimal therapeutic strategies. Simple ‘bedside' biomarkers could be used in all phases of patient management to avoid unnecessary investigation and guide further management. The focal adhesion complex(FAC) has been implicated in the pathogenesis of multiple inflammatory diseases, including IBD, rheumatoid arthritis, and multiple sclerosis. Utilizing omics technologies has proven to be an efficient approach to identify biomarkers from within the FAC in the field of cancer medicine. Predictive biomarkers are paving the way for the success of precision medicine for cancer patients, but inflammatory diseases have lagged behind in this respect. This review explores the current status of biomarker prediction for inflammatory diseases from within the FAC using omics technologies and highlights the benefits of future potential biomarker identification approaches.     

Matrix vesicles originate from apical membrane microvilli of mineralizing osteoblast‐like Saos‐2 cells

In bone, mineralization is tightly regulated by osteoblasts and hypertrophic chondrocytes which release matrix vesicles (MVs) and control extracellular ionic conditions and matrix composition. MVs are the initial sites of hydroxyapatite (HA) mineral formation. Despite growing knowledge about their morphology and function, their biogenesis is not well understood. The purpose of this work was to determine the source of MVs in osteoblast lineage, Saos‐2 cells, and to check whether MVs originated from microvilli. Microvilli were isolated from the apical plasma membrane of Saos‐2 cells. Their morphology, structure, and function were compared with those of MVs. The role of actin network in MV release was investigated by using microfilament perturbing drugs. When examined by electron microscopy MVs and microvillar vesicles were found to exhibit similar morphology with trilaminar membranes and diameters in the same range. Both types of vesicles were able to induce HA formation. Their electrophoretic profiles displayed analogous enrichment in alkaline phosphatase, Na⁺/K⁺ ATPase, and annexins A2 and A6. MVs and microvillar vesicles exhibited almost the same lipid composition with a higher content of cholesterol, sphingomyelin, and phosphatidylserine as compared to plasma membrane. Finally, cytochalasin D, which inhibits actin polymerization, was found to stimulate release of MVs. Our findings were consistent with the hypothesis that MVs originated from cell microvilli and that actin filament disassembly was involved in their biogenesis. J. Cell. Biochem. 106: 127–138, 2009. © 2008 Wiley‐Liss, Inc.     

Niche origin of mesenchymal stem cells derived microvesicles determines opposing effects on NSCLC: Primary versus metastatic

Novel therapeutic approaches that address the malignant cells in their stroma microenvironment are urgently needed in lung cancer. The stroma resident mesenchymal stem cells (MSCs) interact with cancer cells in diverse ways including microvesicles (MVs) that transfer proteins and RNA species thereby modulating recipient cells' phenotype. Previously, we have demonstrated that MSCs' secretome from the primary non-small cell lung cancer (NSCLC) niche (lung) and metastatic niche (bone marrow (BM)) demonstrate opposite effects on NSCLC cells in a translation initiation (TI) dependent manner. Here, we examined the effect of MVs secreted from BM-MSCs' or lung-MSCs (healthy, NSCLC) to NSCLC phenotype. Briefly, NSCLC cell lines treated with Lung or BM-MSCs' MVs were assayed for viability (WST-1), cell count/death (trypan), migration (scratch), TI status and MAPKs activation (immunoblotting). Corresponding to previous published trends, Lung-MSCs' MVs promoted NSCLC cells' assayed traits whereas, BM-MSCs' MVs suppressed them. Activation of MAPKs and autophagy was registered in lung-MSCs MVs treated NSCLC cell lines only. Furthermore, lung-MSCs' MVs' treated NSCLC cells demonstrated an early (5 min) activation of MAPKs and TI factors (peIF4E/peIF4GI) not evident in BM-MSCs MVs treated cells. These observations depict a role for MSCs'-MVs in NSCLC phenotype design and display distinct differences between the primary and metastatic niches that correspond to disease progression.In conclusion, the systemic nature of MVs marks them as attractive therapeutic markers/targets and we propose that identification of specific cargoes/signals that differentiate between MSCs MVs of primary and metastatic niches may introduce fresh therapeutic approaches.     

Nonfunctioning Adrenal Incidentalomas are not Clinically Silent: A Longitudinal Cohort Study

Objective: The association between nonfunctioning adrenal incidentalomas (NFAIs) and cardiometabolic diseases remains controversial. This retrospective cohort study investigated whether NFAIs are related with prevalent and incident cardiometabolic diseases.Methods: This study included 154 patients with biochemically confirmed NFAIs and 1:3 age and sex-matched controls without adrenal incidentalomas (n = 462) among subjects who underwent abdominal computed tomography at a single healthcare center in 2003–2012. Electronic medical records were reviewed for comorbidities at baseline and during a mean follow-up of 7.5 years. The logistic regression analysis for prevalent cardiometabolic diseases and the survival analysis for incident cardiometabolic diseases were performed.Results: The subjects were 55.7 ± 8.8 years of age and predominantly male (73.1%). The NFAI group had a higher body mass index compared to the age and sex-matched control group (25.1 ± 2.8 vs. 24.0 ± 2.8 kg/m²; P<.001). In a cross-sectional design, covariate-adjusted logistic regression showed significantly higher odds ratios (ORs) for diabetes mellitus and hypertension in the NFAI group (adjusted OR [95% confidence interval [CI]], 1.89 [1.17 to 3.06] and 2.26 [1.47 to 3.50], respectively). The NFAI group had a 2-fold higher risk of insulin resistance (adjusted ORs [95% CI], 2.03 [1.06 to 3.90]). Moreover, NFAI subjects with diabetes mellitus had a greater increase in size of adrenal lesions than those without diabetes mellitus (3.4 ± 5.5 vs. 1.4 ± 5.5 mm; P =.048). However, in the survival analysis, the incidence of any cardiometabolic diseases did not differ between the NFAI and control groups.Conclusion: NFAIs are related to prevalent diabetes mellitus or hypertension in our cross-sectional study. However, the presence of NFAIs did not affect the development of cardiometabolic diseases.Abbreviations: ACTH = adrenocorticotropic hormone; AI = adrenal incidentaloma; BMI = body mass index; CI = confidence interval; CT = computed tomography; HbA1c = hemoglobin A1c; HOMA-IR = homeostasis model assessment of insulin resistance; HU = Hounsfield units; MACE = mild autonomous cortisol excess; NFAI = nonfunctioning adrenal incidentaloma; OR = odds ratio     

Chemical constituents isolated from Disporum viridescens leaves and their inhibitory effect on nitric oxide production in BV2 microglial cells

Excessive NO (nitric oxide) has been associated with the pathogenesis of various neurodegenerative diseases including Alzheimer’s disease (AD). In our screening system using LPS-activated BV2 microglial cells, the methanolic extract of Disporum viridescens leaves was found to have significant NO inhibitory activity. Bioactivity-guided isolation yielded a new phenylpropanoid characterized as 4-ally-2,6-dimethoxyphenyl 1-O-β-d-apiofuranosyl (1 → 6)-β-d-glucopyranoside (12) with 21 known compounds from the leaves of D. viridescens. Among them, compounds 2 and 4 significantly inhibited NO production. Thus, we further elucidated the anti-inflammatory mechanism of these lignans. Especially, compound 4 inhibited the expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) through the suppression of the MAPK signaling pathway. Taken together, the anti-inflammatory activities of the active constituents isolated from D. viridescens leaves could have therapeutic potential against neurodegenerative diseases.     

The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands

One of the recent breakthroughs in stem cell research has been the reprogramming of human somatic cells to an embryonic stem cell (ESC)-like state (induced pluripotent stem cells, iPS cells). Similar to ESCs, iPS cells can differentiate into derivatives of the three germ layers, for example cardiomyocytes, pancreatic cells or neurons. This technique offers a new approach to investigating disease pathogenesis and to the development of novel therapies. It may now be possible to generate iPS cells from somatic cells of patients who suffer from vascular genetic diseases, such as hereditary haemorrhagic telangiectasia (HHT). The iPS cells will have a similar genotype to that of the patient and can be differentiated in vitro into the cell type(s) that are affected in the patient. Thus they will serve as excellent models for a better understanding of mechanisms underlying the disease. This, together with the ability to test new drugs, could potentially lead to novel therapeutic concepts in the near future. Here we report the first derivation of three human iPS cell lines from two healthy individuals and one HHT patient in the Netherlands. The iPS cells resembled ESCs in morphology and expressed typical ESC markers. In vitro, iPS cells could be differentiated into cells of the three germ layers, including beating cardiomyocytes and vascular cells. With this technique it will be possible to establish human cardiovascular disease models from patient biopsies provided by the principal hospitals in the Netherlands. (Neth Heart J 2010;18:51-4.)     

MosaicBase:A Knowledgebase of Postzygotic Mosaic Variants in Noncancer Disease-related and Healthy Human Individuals

Mosaic variants resulting from postzygotic mutations are prevalent in the human genome and play important roles in human diseases. However, except for cancer-related variants, there is no collection of postzygotic mosaic variants in noncancer disease-related and healthy individuals. Here, we present MosaicBase, a comprehensive database that includes 6698 mosaic variants related to 266 noncancer diseases and 27,991 mosaic variants identified in 422 healthy individuals. Genomic and phenotypic information of each variant was manually extracted and curated from 383 publications. MosaicBase supports the query of variants with Online Mendelian Inheritance in Man (OMIM) entries, genomic coordinates, gene symbols, or Entrez IDs. We also provide an integrated genome browser for users to easily access mosaic variants and their related annotations for any genomic region. By analyzing the variants collected in MosaicBase, we find that mosaic variants that directly contribute to disease phenotype show features distinct from those of variants in individuals with mild or no phenotypes, in terms of their genomic distribution, mutation signatures, and fraction of mutant cells. MosaicBase will not only assist clinicians in genetic counseling and diagnosis but also provide a useful resource to understand the genomic baseline of postzygotic mutations in the general human population. MosaicBase is publicly available at http://mosaicbase.com/ or http://49.4.21.8:8000.