Proteomic Analysis of Mesenchymal Stromal Cell-Derived Extracellular Vesicles and Reconstructed Membrane Particles

Extracellular vesicles (EV) derived from mesenchymal stromal cells (MSC) are a potential therapy for immunological and degenerative diseases. However, large-scale production of EV free from contamination by soluble proteins is a major challenge. The generation of particles from isolated membranes of MSC, membrane particles (MP), may be an alternative to EV. In the present study we generated MP from the membranes of lysed MSC after removal of the nuclei. The yield of MP per MSC was 1 × 105 times higher than EV derived from the same number of MSC. To compare the proteome of MP and EV, proteomic analysis of MP and EV was performed. MP contained over 20 times more proteins than EV. The proteins present in MP evidenced a multi-organelle origin of MP. The projected function of the proteins in EV and MP was very different. Whilst proteins in EV mainly play a role in extracellular matrix organization, proteins in MP were interconnected in diverse molecular pathways, including protein synthesis and degradation pathways and demonstrated enzymatic activity. Treatment of MSC with IFNγ led to a profound effect on the protein make up of EV and MP, demonstrating the possibility to modify the phenotype of EV and MP through modification of parent MSC. These results demonstrate that MP are an attractive alternative to EV for the development of potential therapies. Functional studies will have to demonstrate therapeutic efficacy of MP in preclinical disease models.

Lipid Polymorphism of the Subchloroplast—Granum and Stroma Thylakoid Membrane–Particles. II. Structure and Functions

In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments—in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering—but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.

Effect of respiratory inhibitors and quinone analogues on the aerobic electron transport system of Eikenella corrodens

The effects of respiratory inhibitors, quinone analogues and artificial substrates on the membrane-bound electron transport system of the fastidious β-proteobacteriumEikenella corrodensgrown under O2-limited conditions were studied. NADH respiration in isolated membrane particles were partially inhibited by rotenone, dicoumarol, quinacrine, flavone, and capsaicin. A similar response was obtained when succinate oxidation was performed in the presence of thenoyltrifluoroacetone and N,N’-dicyclohexylcarbodiimide. NADH respiration was resistant to site II inhibitors and cyanide, indicating that a percentage of the electrons transported can reach O2without thebc1complex. Succinate respiration was sensitive to myxothiazol, antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Juglone, plumbagin and menadione had higher reactivity with NADH dehydrogenase. The membrane particles showed the highest oxidase activities with ascorbate-TCHQ (tetrachlorohydroquinone), TCHQ alone, and NADH-TMPD (N,N,N’,N’-tetramethyl-p-phenylenediamine), and minor activity levels with ascorbate-DCPIP (2,6-dichloro-phenolindophenol) and NADH-DCPIP. The substrates NADH-DCPIP, NADH-TMPD and TCHQ were electron donors to cyanide-sensitivecbb'cytochromecoxidase. The presence of dissimilatory nitrate reductase in the aerobic respiratory system ofE.corrodensATCC 23834 was demonstrated by first time. Our results indicate that complexes I and II have resistance to their classic inhibitors, that the oxidation of NADH is stimulated by juglone, plumbagin and menadione, and that sensitivity to KCN is stimulated by the substrates TCHQ, NADH-DCPIP and NADH-TMPD.

Membrane Particles Derived From Adipose Tissue Mesenchymal Stromal Cells Improve Endothelial Cell Barrier Integrity

Proinflammatory stimuli lead to endothelial injury, which results in pathologies such as cardiovascular diseases, autoimmune diseases, and contributes to alloimmune responses after organ transplantation. Both mesenchymal stromal cells (MSC) and the extracellular vesicles (EV) released by them are widely studied as regenerative therapy for the endothelium. However, for therapeutic application, the manipulation of living MSC and large-scale production of EV are major challenges. Membrane particles (MP) generated from MSC may be an alternative to the use of whole MSC or EV. MP are nanovesicles artificially generated from the membranes of MSC and possess some of the therapeutic properties of MSC. In the present study we investigated whether MP conserve the beneficial MSC effects on endothelial cell repair processes under inflammatory conditions. MP were generated by hypotonic shock and extrusion of MSC membranes. The average size of MP was 120 nm, and they showed a spherical shape. The effects of two ratios of MP (50,000; 100,000 MP per target cell) on human umbilical vein endothelial cells (HUVEC) were tested in a model of inflammation induced by TNFα. Confocal microscopy and flow cytometry showed that within 24 hours >90% of HUVEC had taken up MP. Moreover, MP ended up in the lysosomes of the HUVEC. In a co-culture system of monocytes and TNFα activated HUVEC, MP did not affect monocyte adherence to HUVEC, but reduced the transmigration of monocytes across the endothelial layer from 138 ± 61 monocytes per microscopic field in TNFα activated HUVEC to 61 ± 45 monocytes. TNFα stimulation induced a 2-fold increase in the permeability of the HUVEC monolayer measured by the translocation of FITC-dextran to the lower compartment of a transwell system. At a dose of 1:100,000 MP significantly decreased endothelial permeability (1.5-fold) respect to TNFα Stimulated HUVEC. Finally, MP enhanced the angiogenic potential of HUVEC in an in vitro Matrigel assay by stimulating the formation of angiogenic structures, such as percentage of covered area, total tube length, total branching points, total loops. In conclusion, MP show regenerative effects on endothelial cells, opening a new avenue for treatment of vascular diseases where inflammatory processes damage the endothelium.

Mesenchymal Stromal Cell Derived Membrane Particles Are Internalized by Macrophages and Endothelial Cells Through Receptor-Mediated Endocytosis and Phagocytosis

Mesenchymal stromal cells (MSC) are a promising therapy for inflammatory diseases. However, MSC are large and become trapped in the lungs after intravenous infusion, where they have a short survival time. To steer MSC immunoregulatory therapy beyond the lungs, we generated nm-sized particles from MSC membranes (membrane particles, MP), which have immunomodulatory properties, and investigated their internalization and mode of interaction in macrophages subtypes and human umbilical vein endothelial cells (HUVEC) under control and inflammatory conditions. We found that macrophages and HUVEC take up MP in a dose, time, and temperature-dependent manner. Specific inhibitors for endocytotic pathways revealed that MP internalization depends on heparan sulfate proteoglycan-, dynamin-, and clathrin-mediated endocytosis but does not involve caveolin-mediated endocytosis. MP uptake also involved the actin cytoskeleton and phosphoinositide 3-kinase, which are implicated in macropinocytosis and phagocytosis. Anti-inflammatory M2 macrophages take up more MP than pro-inflammatory M1 macrophages. In contrast, inflammatory conditions did not affect the MP uptake by HUVEC. Moreover, MP induced both anti- and pro-inflammatory responses in macrophages and HUVEC by affecting gene expression and cell surface proteins. Our findings on the mechanisms of uptake of MP under different conditions help the development of target-cell specific MP therapy to modulate immune responses.

Plasmonic Nanobowtiefluidic Device for Sensitive Detection of Glioma Extracellular Vesicles by Raman Spectrometry

Cancer cells shed into biofluids extracellular vesicles (EVs) — nanoscale membrane particles carrying diagnostic information. EVs shed by heterogeneous populations of tumor cells offer a unique opportunity to access biologically...