Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation

Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte

Finger beat-to-beat blood pressure responses to successive hand elevations.

We investigated finger beat-to-beat blood pressure responses to a series of successive hand elevations in 14 normal volunteers. By passive elevation of the hand by 40 cm and lowering it again after a minute, calibrated hydrostatic pressure changes were induced in the finger arteries of the subjects. Three successive procedures with a 2-min interval between them were performed. Transitions between positions were completed smoothly over a 10-s period. Non-invasive beat-to-beat mean arterial pressure (MAP) in the finger arteries was measured by applying the servo-oscillometric physiograph (University of Tartu, Estonia). A good agreement between the evoked MAP changes during all the three hand elevations (-31.2, -30.4 and -30.0 mmHg, respectively) and the calculated hydrostatic pressure change (-31.0 mmHg) was obtained. The height difference of approximately 40 cm and rate of 4-5 cm/s can be recommended for the hand elevation test, greater postural changes and higher rates may diminish agreement between the measured blood pressure response and the corresponding hydrostatic pressure change. The applied hydrostatic test may be helpful for assessing the accuracy of beat-to-beat finger blood pressure measurement

The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development

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Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay.

Cell-penetrating peptides (CPPs) have shown great potency for cargo delivery both in vitro and in vivo. Different biologically relevant molecules need to be delivered into appropriate cellular compartments in order to be active, for instance certain drugs/molecules, e.g. antisense oligonucleotides, peptides, and cytotoxic agents require delivery into the cytoplasm. Assessing uptake mechanisms of CPPs can help to develop novel and more potent cellular delivery vectors, especially in cases when reaching a specific intracellular target requires involvement of a specific internalization pathway. Here we measure the overall uptake kinetics, with emphasis on cytoplasmic delivery, of three cell-penetrating peptides M918, TP10 and pVec using a quenched fluorescence assay. We show that both the uptake levels and kinetic constants depend on the endocytosis inhibitors used in the experiments. In addition, in some cases only the internalization rate is affected by the endocytosis inhibitors while the total uptake level is not and vice versa, which emphasizes importance of kinetic studies when assessing the uptake mechanisms of CPPs. Also, there seems to be a correlation between lower total cellular uptake and higher first-order rate constants. Furthermore, this may indicate simultaneous involvement of different endocytic pathways with different efficacies in the internalization process, as hypothesized but not shown earlier in an uptake kinetics assay

Prediction of Cell-Penetrating Peptides Using Artificial Neural Networks.

An investigation of cell-penetrating peptides (CPPs) by using combination of Artificial Neural Networks (ANN) and Principle Component Analysis (PCA) revealed that the penetration capability (penetrating/non-penetrating) of 101 examined peptides can be predicted with accuracy of 80%-100%. The inputs of the ANN are the main characteristics classifying the penetration. These molecular characteristics (descriptors) were calculated for each peptide and they provide bio-chemical insights for the criteria of penetration. Deeper analysis of the PCA results also showed clear clusterization of the peptides according to their molecular features

PepFect14 peptide vector for efficient gene delivery in cell cultures.

The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130-170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery

CSF extracellular vesicle proteomics demonstrates altered protein homeostasis in amyotrophic lateral sclerosis

Background Extracellular vesicles (EVs) released by neurons and glia reach the cerebrospinal fluid (CSF). Studying the proteome of CSF-derived EVs offers a novel perspective on the key intracellular processes associated with the pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and a potential source from which to develop biomarkers. Methods CSF EVs were extracted using ultrafiltration liquid chromatography from ALS patients and controls. EV size distribution and concentration was measured using nanoparticle tracking analysis and liquid chromatography-tandem mass spectrometry proteomic analysis performed. Results CSF EV concentration and size distribution did not differ between ALS and control groups, nor between a sub-group of ALS patients with or without an associated hexanucleotide repeat expansion (HRE) in C9orf72. Univariate proteomic analysis identified downregulation of the pentameric proteasome-like protein Bleomycin hydrolase in ALS patients, whilst Gene Ontology enrichment analysis demonstrated downregulation of proteasome core complex proteins (8/8 proteins, normalized enrichment ratio -1.77, FDR-adjusted p = 0.057) in the ALS group. The sub-group of ALS patients associated with the C9orf72 HRE showed upregulation in Ubiquitin-like modifying-activating protein 1 (UBA1) compared to non-C9orf72 cases. Conclusions Proteomic analysis of CSF EVs in ALS detects intracellular alterations in protein homeostatic mechanisms, previously only identified in pathological tissues. This supports the wider use of CSF EVs as a source of novel biomarkers reflecting key and potentially druggable pathological intracellular pathway alterations in ALS.</p