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1.
Small ; 17(5): e2006786, 2021 02.
Article in English | MEDLINE | ID: mdl-33448084

ABSTRACT

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.


Subject(s)
Endothelial Cells , Extracellular Vesicles , Cluster Analysis , Humans , Plasma , Unsupervised Machine Learning
2.
Cells ; 9(2)2020 01 28.
Article in English | MEDLINE | ID: mdl-32012900

ABSTRACT

Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis.


Subject(s)
Dental Pulp/cytology , Extracellular Vesicles/metabolism , Mesenchymal Stem Cells/metabolism , Neovascularization, Physiologic , Adolescent , Angiogenesis Inducing Agents/metabolism , Animals , Chemotactic Factors/pharmacology , Chickens , Endocytosis/drug effects , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Extracellular Vesicles/drug effects , Extracellular Vesicles/ultrastructure , Female , Humans , Male , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/ultrastructure , Neovascularization, Physiologic/drug effects , Paracrine Communication/drug effects , Time Factors , Young Adult
3.
Front Immunol ; 9: 1789, 2018.
Article in English | MEDLINE | ID: mdl-30131806

ABSTRACT

Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-α induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) in vitro. Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-α as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes.


Subject(s)
Endothelial Cells/metabolism , Extracellular Vesicles/metabolism , Inflammation Mediators/metabolism , Monocytes/metabolism , Chemokines/metabolism , Cytokines/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Tumor Necrosis Factor-alpha/metabolism
4.
Chem Commun (Camb) ; 51(87): 15858-15861, 2015 Nov 11.
Article in English | MEDLINE | ID: mdl-26377628

ABSTRACT

Functional nanocarriers were synthesized using an in situ inverse miniemulsion polymerization employing thiol-isocyanate reactions at the droplet interface to encapsulate hydrophilic payloads. The morphology of the nanocarriers is conveniently tunable by varying the reaction conditions and the dispersions are easily transferable to the aqueous phase.


Subject(s)
Drug Delivery Systems , Isocyanates/chemistry , Nanoparticles/chemistry , Sulfhydryl Compounds/chemistry , Antibiotics, Antineoplastic/chemistry , Doxorubicin/chemistry , Emulsions , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Particle Size , Potassium Chloride/chemistry , Toluene 2,4-Diisocyanate/chemistry , Urethane/chemical synthesis
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