The effects of mesenchymal stem cell (MSC)‐derived extracellular vesicles were compared with those of MSCs i.v. delivered 1, 3, and 5 days or 1 day after focal cerebral ischemia in mice. Motor ...coordination deficits, brain injury, immune responses in peripheral blood and brain, and cerebral angiogenesis and neurogenesis were analyzed. Postischemic immunosuppression was attenuated in peripheral blood 6 days after ischemia, providing an appropriate external milieu for successful brain remodeling.
Although the initial concepts of stem cell therapy aimed at replacing lost tissue, more recent evidence has suggested that stem and progenitor cells alike promote postischemic neurological recovery by secreted factors that restore the injured brain's capacity to reshape. Specifically, extracellular vesicles (EVs) derived from stem cells such as exosomes have recently been suggested to mediate restorative stem cell effects. In order to define whether EVs indeed improve postischemic neurological impairment and brain remodeling, we systematically compared the effects of mesenchymal stem cell (MSC)‐derived EVs (MSC‐EVs) with MSCs that were i.v. delivered to mice on days 1, 3, and 5 (MSC‐EVs) or on day 1 (MSCs) after focal cerebral ischemia in C57BL6 mice. For as long as 28 days after stroke, motor coordination deficits, histological brain injury, immune responses in the peripheral blood and brain, and cerebral angiogenesis and neurogenesis were analyzed. Improved neurological impairment and long‐term neuroprotection associated with enhanced angioneurogenesis were noticed in stroke mice receiving EVs from two different bone marrow‐derived MSC lineages. MSC‐EV administration closely resembled responses to MSCs and persisted throughout the observation period. Although cerebral immune cell infiltration was not affected by MSC‐EVs, postischemic immunosuppression (i.e., B‐cell, natural killer cell, and T‐cell lymphopenia) was attenuated in the peripheral blood at 6 days after ischemia, providing an appropriate external milieu for successful brain remodeling. Because MSC‐EVs have recently been shown to be apparently safe in humans, the present study provides clinically relevant evidence warranting rapid proof‐of‐concept studies in stroke patients.
Significance
Transplantation of mesenchymal stem cells (MSCs) offers an interesting adjuvant approach next to thrombolysis for treatment of ischemic stroke. However, MSCs are not integrated into residing neural networks but act indirectly, inducing neuroprotection and promoting neuroregeneration. Although the mechanisms by which MSCs act are still elusive, recent evidence has suggested that extracellular vesicles (EVs) might be responsible for MSC‐induced effects under physiological and pathological conditions. The present study has demonstrated that EVs are not inferior to MSCs in a rodent stroke model. EVs induce long‐term neuroprotection, promote neuroregeneration and neurological recovery, and modulate peripheral post‐stroke immune responses. Also, because EVs are well‐tolerated in humans, as previously reported, the administration of EVs under clinical settings might set the path for a novel and innovative therapeutic stroke concept without the putative side effects attached to stem cell transplantation.
Preterm neonates are susceptible to perinatal hypoxic-ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic-ischemic brain injury in ovine fetuses, we ...have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) in a preclinical model of preterm hypoxic-ischemic brain injury. Ovine fetuses were subjected to global hypoxia-ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC-EVs. The therapeutic effects of MSC-EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC-EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC-EV treatment. Our data demonstrate that MSC-EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic-ischemic injury of the preterm brain. Our study results suggest that a cell-free preparation comprising neuroprotective MSC-EVs could substitute MSCs in the treatment of preterm neonates with hypoxic-ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells.
Bone marrow-derived mesenchymal stromal cells (MSCs) show promise in treating hypoxic-ischemic injury of the preterm brain. Study results suggest administration of extracellular vesicles, rather than intact MSCs, is sufficient to exert therapeutic effects and avoids potential concerns associated with administration of living cells. The therapeutic efficacy of systemically administered mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) on hypoxia-ischemia-induced injury was assessed in the preterm ovine brain. Impaired function and structural injury of the fetal brain was improved following global hypoxia-ischemia. A cell-free preparation of MSC-EVs could substitute for the cellular counterpart in the treatment of preterm neonates with hypoxic-ischemic brain injury. This may open new clinical applications for "off-the-shelf" interventions with MSC-EVs.
Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. ...Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin‐1 and galectin‐3, we identified several interacting pairs, such as CXCL12 and galectin‐3. Based on NMR and MD studies of the CXCL12/galectin‐3 heterodimer, we identified contact sites between CXCL12 β‐strand 1 and Gal‐3 F‐face residues. Mutagenesis of galectin‐3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin‐3, but not its mutants, inhibited CXCL12‐induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin‐3 attenuated CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted.
Synopsis
Chemokines and galectins are simultaneously upregulated during inflammation and mediate leukocyte recruitment. A systematic screen now demonstrates their physical interaction as heterodimers, identifying several novel interacting pairs.
Chemokines and galectins can engage in cross talk by pairing, as exemplified by galectin‐3 and CXCL12.
The association of CXCL12 with galectin‐3 appears to have potential for modulating chemokine activity.
Galectin‐3 inhibits CXCL12‐induced chemotaxis of leukocytes and their recruitment to inflammation sites.
Galectin‐3 attenuates CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex.
Chemokines and galectins are simultaneously upregulated during inflammation and mediate leukocyte recruitment. A systematic screen now demonstrates their physical interaction as heterodimers, identifying several novel interacting pairs.
Recognition of glycans by lectins is emerging as (patho)physiologically broadly used mode of cellular information transfer. Whereas the direct ligand-receptor contact is often already thoroughly ...characterized, the functional relevance of aspects of architecture such as modular design and valence of lectins is less well defined.
Following an introduction to modular lectin design, three levels of methodology are then reviewed that delineate lectin structure-activity relationships beyond glycan binding, with emphasis on domain shuffling.
Engineering of variants by modular transplantation facilitates versatile Nature-inspired design switches and access to new combinations with translational potential, as exemplified for human adhesion/growth-regulatory galectins.
To gain an understanding of the functional significance of natural variations in quaternary structure and modular design within a protein family is a current challenge. Strategic application of methods of the described phases is a means to respond to this challenge.
•Lectins have distinct types of design of not yet known functional significance.•Chemical modifications are a means to alter quaternary structure and cross-linking.•Site-specific sequence changes generate lectins with new specificity.•Modular transplantations generate lectin variants with new design and function.•Human lectin variants have innovative potential for therapy.
Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type ...specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.
The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to ...the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic α-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 α-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of α-dystroglycanopathy.
Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. ...Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectinmediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3′-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity.
Exosomes are small membrane vesicles, which eukaryotic cells secrete into their extracellular environment. They are formed as intraluminal vesicles by inward budding of the limiting membrane into the ...lumen of late endosomes. Upon fusion of thus arising multivesicular bodies with the plasma membrane, these vesicles are released as exosomes and enter body fluids such as blood plasma, urine and saliva. Containing certain combinations of lipids, adhesion and intercellular signaling molecules as well as RNAs, exosomes participate in intercellular communication processes. Depending on their origin, exosomes can modulate immune-regulatory processes, set up tumor escape mechanisms and mediate regenerative or degenerative processes, amongst others. In summary, exosomes are molecular complex intercellular signaling organelles with multiple functions, which appear as promising new tools for the clinical diagnostics and potentially for novel therapeutic strategies.
Chemical and biological tools are harnessed to investigate the impact of spatial factors for functional pairing of human lectins with counterreceptors. The homodimeric adhesion/growth‐regulatory ...galectin‐1 and a set of covalently linked homo‐oligomers from di‐ to tetramers serve as proof‐of‐principle test cases. Glycodendrimersomes provide a versatile and sensitive diagnostic platform to reveal thresholds for ligand density and protein concentration in aggregation assays (trans‐activity), irrespective of linker length between lectin domains. Monitoring the affinity of cell binding and ensuing tumor growth inhibition reveal the linker length to be a bidirectional switch for cis‐activity. The discovery that two aspects of lectin functionality (trans‐ versus cis‐activity) respond non‐uniformly to a structural change underscores the power of combining synthetic and biological tools to advance understanding of the sugar functionality of the cell surface.
Carbohydrate‐directed cell adhesion: Chemical programming of the glycan presentation of glycodendrimersomes (GDSs) and the responsiveness of cells to lectin binding have been combined to define the structure–activity relationships for trans‐bridging and cis‐crosslinking of human lectins. Consequences of structural engineering of both lectin and glycan topology are explored and quantified (Gal‐1=galectin‐1).
Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct ...signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N′-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.
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