Cargo Proteins Research Articles

Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal.

During the progression of knee osteoarthritis (OA), the synovium and infrapatellar fat pad (IFP) can serve as source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important pain-transmitting, immune, and inflammation modulating neuropeptides. Our previous studies showed that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo. On this basis, our hypothesis is that CD10-bound IFP-MSC sEVs can be engineered to target CGRP while retaining their anti-inflammatory phenotype. Herein, human IFP-MSC cultures were transduced with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for a CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were isolated and their sEVs' miRNA and protein cargos were assessed using multiplex methods. Our results showed that purified aCGRP IFP-MSC cultures yielded sEVs with cargo of 147 distinct MSC-related miRNAs. Reactome analysis of miRNAs detected in these sEVs revealed strong involvement in the regulation of target genes involved in pathways that control pain, inflammation and cartilage homeostasis. Protein array of the sEVs cargo demonstrated high presence of key immunomodulatory and reparative proteins. Stimulated macrophages exposed to aCGRP IFP-MSC sEVs demonstrated a switch towards an alternate M2 status. Also, stimulated cortical neurons exposed to aCGRP IFP-MSC sEVs modulate their molecular pain signaling profile. Collectively, our data suggest that yielded sEVs can putatively target CGRP in vivo, while containing potent anti-inflammatory and analgesic cargo, suggesting the promise for novel sEVs-based therapeutic approaches to diseases such as OA.

Proteomic Profiling of Plasma- and Gut-Derived Extracellular Vesicles in Obesity.

Obesity entails metabolic alterations across multiple organs, highlighting the role of inter-organ communication in its pathogenesis. Extracellular vesicles (EVs) are communication agents in physiological and pathological conditions, and although they have been associated with obesity comorbidities, their protein cargo in this context remains largely unknown. To decipher the messages encapsulated in EVs, we isolated plasma-derived EVs from a diet-induced obese murine model. Obese plasma EVs exhibited a decline in protein diversity while control EVs revealed significant enrichment in protein-folding functions, highlighting the importance of proper folding in maintaining metabolic homeostasis. Previously, we revealed that gut-derived EVs' proteome holds particular significance in obesity. Here, we compared plasma and gut EVs and identified four proteins exclusively present in the control state of both EVs, revealing the potential for a non-invasive assessment of gut health by analyzing blood-derived EVs. Given the relevance of post-translational modifications (PTMs), we observed a shift in chromatin-related proteins from glycation to acetylation in obese gut EVs, suggesting a regulatory mechanism targeting DNA transcription during obesity. This study provides valuable insights into novel roles of EVs and protein PTMs in the intricate mechanisms underlying obesity, shedding light on potential biomarkers and pathways for future research.

RANBP17 Overexpression Restores Nucleocytoplasmic Transport and Ameliorates Neurodevelopment in Induced DYT1 Dystonia Motor Neurons.

DYT1 dystonia is a debilitating neurological movement disorder, and it represents the most frequent and severe form of hereditary primary dystonia. There is currently no cure for this disease due to its unclear pathogenesis. In our previous study utilizing patient-specific motor neurons (MNs), we identified distinct cellular deficits associated with the disease, including a deformed nucleus, disrupted neurodevelopment, and compromised nucleocytoplasmic transport (NCT) functions. However, the precise molecular mechanisms underlying these cellular impairments have remained elusive. In this study, we revealed the genome-wide changes in gene expression in DYT1 MNs through transcriptomic analysis. We found that those dysregulated genes are intricately involved in neurodevelopment and various biological processes. Interestingly, we identified that the expression level of RANBP17, a RAN-binding protein crucial for NCT regulation, exhibited a significant reduction in DYT1 MNs. By manipulating RANBP17 expression, we further demonstrated that RANBP17 plays an important role in facilitating the nuclear transport of both protein and transcript cargos in induced human neurons. Excitingly, the overexpression of RANBP17 emerged as a substantial mitigating factor, effectively restoring impaired NCT activity and rescuing neurodevelopmental deficits observed in DYT1 MNs. These findings shed light on the intricate molecular underpinnings of impaired NCT in DYT1 neurons and provide novel insights into the pathophysiology of DYT1 dystonia, potentially leading to the development of innovative treatment strategies.

Formation of HopQ1:14-3-3 complex in the host cytoplasm modulates nuclear import rate of Pseudomonas syringae effector in Nicotiana benthamiana cells.

HopQ1, a type three effector from Pseudomonas syringae upon phosphorylation coopts plant 14-3-3 proteins to control its stability and subcellular localization. Mass spectrometry of the cytoplasm-restricted effector revealed that HopQ1 already in this subcellular compartment undergoes phosphorylation at serine 51 within the canonical 14-3-3 binding motif and within the second putative 14-3-3 binding site, 24RTPSES29. Our analyses revealed that the stoichiometry of the HopQ1:14-3-3a complex is 1:2 indicating that both binding sites of HopQ1 are involved in the interaction. Notably, 24RTPSES29 comprises a putative nuclear translocation signal (NTS). Although a peptide containing NTS mediates nuclear import of a Cargo protein suggesting its role in the nuclear trafficking of HopQ1, a deletion of 25TPS27 does not change HopQ1 distribution. In contrast, elimination of 14-3-3 binding site, accelerates nuclear trafficking the effector. Collectively, we show that formation of the HopQ1:14-3-3 complex occurs in the host cytoplasm and slows down the effector translocation into the nucleus. These results provide a mechanism that maintains the proper nucleocytoplasmic partitioning of HopQ1, and at the same time is responsible for the relocation of 14-3-3s from the nucleus to cytoplasm in the presence of the effector.

Controlled Refolding of Denatured IL-12 Using In Situ Antigen-Capturing Nanochaperone Remarkably Reduces the Systemic Toxicity and Enhances Cancer Immunotherapy.

Cytokines are powerful in cancer immunotherapy, however, their therapeutic potential is limited by the severe systemic toxicity. Here a potent strategy to reduce the toxicity of systemic cytokine therapy by delivering its denatured form using a finely designed nanochaperone, is described. It is demonstrated that even if the denatured protein cargos are occasionally released under normal physiological conditions they are still misfolded, while can effectively refold into native states and release to function in tumor microenvironment. Consequently, the systemic toxicity of cytokines is nearly completely overcome. Moreover, an immunogenic cell death (ICD)-inducing chemotherapeutic is further loaded and delivered to tumor using this nanochaperone to trigger the release of tumor-associated antigens (TAAs) that are subsequently captured in situ by nanochaperone and then reflows into lymph nodes (LNs) to promote antigen cross-presentation. This optimized personalized nanochaperone-vaccine demonstrates unprecedented suppressive effects against large, advanced tumors, and in combination with immune checkpoint blockade (ICB) therapy results in a significant abscopal effect and inhibition of postoperative tumor recurrence and metastasis. Hence, this approach provides a simple and universal delivery strategy to reduce the systemic toxicities of cytokines, as well as provides a robust personalized cancer vaccination platform, which may find wide applications in cancer immunotherapy.

Exploring the Potential of Exosomes as Biomarkers in Tuberculosis and Other Diseases.

Tuberculosis (TB) is a major cause of morbidity and mortality and remains an important public health issue in developing countries worldwide. The existing methods and techniques available for the diagnosis of TB are based on combinations of laboratory (chemical and biological), radiological, and clinical tests. These methods are sophisticated and laborious and have limitations in terms of sensitivity, specificity, and accuracy. Clinical settings need improved diagnostic biomarkers to accurately detect biological changes due to pathogen invasion and pharmacological responses. Exosomes are membrane-bound vesicles and mediators of intercellular signaling processes that play a significant role in the pathogenesis of various diseases, such as tuberculosis, and can act as promising biomarkers for the monitoring of TB infection. Compared to conventional biomarkers, exosome-derived biomarkers are advantageous because they are easier to detect in different biofluids, are more sensitive and specific, and may be useful in tracking patients' reactions to therapy. This review provides insights into the types of biomarkers, methods of exosome isolation, and roles of the cargo (proteins) present in exosomes isolated from patients through omics studies, such as proteomics. These findings will aid in developing new prognostic and diagnostic biomarkers and could lead to the identification of new therapeutic targets in the clinical setting.

New aspects of a small GTPase RAB35 in brain development and function.

In eukaryotic cells, organelles in the secretory, lysosomal, and endocytic pathways actively exchange biological materials with each other through intracellular membrane trafficking, which is the process of transporting the cargo of proteins, lipids, and other molecules to appropriate compartments via transport vesicles or intermediates. These processes are strictly regulated by various small GTPases such as the RAS-like in rat brain (RAB) protein family, which is the largest subfamily of the RAS superfamily. Dysfunction of membrane trafficking affects tissue homeostasis and leads to a wide range of diseases, including neurological disorders and neurodegenerative diseases. Therefore, it is important to understand the physiological and pathological roles of RAB proteins in brain function. RAB35, a member of the RAB family, is an evolutionarily conserved protein in metazoans. A wide range of studies using cultured mammalian cells and model organisms have revealed that RAB35 mediates various processes such as cytokinesis, endocytic recycling, actin bundling, and cell migration. RAB35 is also involved in neurite outgrowth and turnover of synaptic vesicles. We generated brain-specific Rab35 knockout mice to study the physiological roles of RAB35 in brain development and function. These mice exhibited defects in anxiety-related behaviors and spatial memory. Strikingly, RAB35 is required for the precise positioning of pyramidal neurons during hippocampal development, and thereby for normal hippocampal lamination. In contrast, layer formation in the cerebral cortex occurred superficially, even in the absence of RAB35, suggesting a predominant role for RAB35 in hippocampal development rather than in cerebral cortex development. Recent studies have suggested an association between RAB35 and neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. In this review, we provide an overview of the current understanding of subcellular functions of RAB35. We also provide insights into the physiological role of RAB35 in mammalian brain development and function, and discuss the involvement of RAB35 dysfunction in neurodegenerative diseases.

Distributing Plant Developmental Regulatory Proteins via Plasmodesmata.

During plant development, mobile proteins, including transcription factors, abundantly serve as messengers between cells to activate transcriptional signaling cascades in distal tissues. These proteins travel from cell to cell via nanoscopic tunnels in the cell wall known as plasmodesmata. Cellular control over this intercellular movement can occur at two likely interdependent levels. It involves regulation at the level of plasmodesmata density and structure as well as at the level of the cargo proteins that traverse these tunnels. In this review, we cover the dynamics of plasmodesmata formation and structure in a developmental context together with recent insights into the mechanisms that may control these aspects. Furthermore, we explore the processes involved in cargo-specific mechanisms that control the transport of proteins via plasmodesmata. Instead of a one-fits-all mechanism, a pluriform repertoire of mechanisms is encountered that controls the intercellular transport of proteins via plasmodesmata to control plant development.

An engineered baculoviral protein and DNA co-delivery system for CRISPR-based mammalian genome editing.

CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.

Mitochondrial derived vesicles- Quo Vadis?

Mitochondria are dynamic, intracellular organelles with a separate genome originating from prokaryotes. They perform numerous functions essential for cellular metabolism and energy production. Mitochondrial-derived vesicles (MDVs) are single or double membrane-enclosed vesicles, formed and released from the mitochondrial sub-compartments into the cytosol, in response to various triggers. MDVs interact with other organelles such as lysosomes and peroxisomes or may be incorporated and excreted via extracellular vesicles (EVs). MDVs selectively incorporate diverse protein and lipid cargoes and are involved in various functions such as mitochondrial quality control, immunomodulation, energy complementation, and compartmentalization and transport. This review aims to provide a summary of the current knowledge of MDVs biogenesis, release, cargoes, and roles.

Ultrastructural and glycoproteomic characterization of Prevotella intermedia: Insights into O-glycosylation and outer membrane vesicles.

Prevotella intermedia, a Gram-negative bacterium from the Bacteroidota phylum, is associated with periodontitis. Other species within this phylum are known to possess the general O-glycosylation system. The O-glycoproteome has been characterized in several species, including Tannerella forsythia, Porphyromonas gingivalis, and Flavobacterium johnsoniae. In our study, we used electron cryotomography (cryoET) and glycoproteomics to reveal the ultrastructure of P. intermedia and characterize its O-glycoproteome. Our cryoET analysis unveiled the ultrastructural details of the cell envelope and outer membrane vesicles (OMVs) of P. intermedia. We observed an electron-dense surface layer surrounding both cells and OMVs. The OMVs were often large (>200 nm) and presented two types, with lumens being either electron-dense or translucent. LC-MS/MS analyses of P. intermedia fractions led to the identification of 1655 proteins, which included 62 predicted T9SS cargo proteins. Within the glycoproteome, we identified 443 unique O-glycosylation sites within 224 glycoproteins. Interestingly, the O-glycosylation motif exhibited a broader range than reported in other species, with O-glycosylation found at D(S/T)(A/I/L/M/T/V/S/C/G/F/N/E/Q/D/P). We identified a single O-glycan with a delta mass of 1531.48 Da. Its sequence was determined by MS2 and MS3 analyses using both collision-induced dissociation and high-energy collisional dissociation fragmentation modes. After partial deglycosylation with trifluoromethanesulfonic acid, the O-glycan sequence was confirmed to be dHex-dHex-HexNAc (HPO3 -C6 H12 O5 )-dHex-Hex-HexA-Hex(dHex). Bioinformatic analyses predicted the localization of O-glycoproteins, with 73 periplasmic proteins, 53 inner membrane proteins, 52 lipoproteins, 26 outer membrane proteins, and 14 proteins secreted by the T9SS.

Effect of Polyphenols on Inflammation Induced by Membrane Vesicles from Staphylococcus aureus.

Staphylococcus aureus, a bacterium found on human skin, produces toxins and various virulence factors that can lead to skin infections such as atopic dermatitis. These toxins and virulence factors are carried in membrane vesicles (MVs), composed of the bacterium's own cell membranes, and are expected to reach host target cells in a concentrated form, inducing inflammation. This study investigated the effects of two polyphenols, (-)-epigallocatechin gallate (EGCG) and nobiletin (NOL), on the expression of S. aureus virulence factors and the inflammation induced by MVs. The study found that EGCG alone decreased the production of Staphylococcal Enterotoxin A (SEA), while both EGCG and NOL reduced biofilm formation and the expression of virulence factor-related genes. When S. aureus was cultured in a broth supplemented with these polyphenols, the resulting MVs showed a reduction in SEA content and several cargo proteins. These MVs also exhibited decreased levels of inflammation-related gene expression in immortalized human keratinocytes. These results suggest that EGCG and NOL are expected to inhibit inflammation in the skin by altering the properties of MVs derived from S. aureus.

Changes in proteomic cargo of circulating extracellular vesicles in response to lifestyle intervention in adolescents with hepatic steatosis

BackgroundRecent studies suggest that proteomic cargo of extracellular vesicles (EVs) may play a role in metabolic improvements following lifestyle interventions. However, the relationship between changes in liver fat and circulating EV-derived protein cargo following intervention remains unexplored. MethodsThe study cohort comprised 18 Latino adolescents with obesity and hepatic steatosis (12 males/6 females; average age 13.3 ±1.2 y) who underwent a six-month lifestyle intervention. EV size distribution and concentration were determined by light scattering intensity; EV protein composition was characterized by liquid chromatography tandem-mass spectrometry. ResultsAverage hepatic fat fraction (HFF) decreased 23% by the end of the intervention (12.5% [5.5] to 9.6% [4.9]; P = 0.0077). Mean EV size was smaller post-intervention compared to baseline (120.2 ± 16.4 nm to 128.4 ± 16.5 nm; P = 0.031), although the difference in mean EV concentration (1.1E+09 ± 4.1E+08 particles/mL to 1.1E+09 ± 1.8E+08 particles/mL; P = 0.656)) remained unchanged. A total of 462 proteins were identified by proteomic analysis of plasma-derived EVs from participants pre- and post- intervention, with 113 proteins showing differential abundance (56 higher and 57 lower) between the two timepoints (adj-p <0.05). Pathway analysis revealed enrichment in complement cascade, initial triggering of complement, creation of C4 and C2 activators, and regulation of complement cascade. Hepatocyte-specific EV affinity purification identified 40 proteins with suggestive (p < 0.05) differential abundance between pre- and post-intervention samples. ConclusionsCirculating EV-derived proteins, particularly those associated with the complement cascade, may contribute to improvements in liver fat in response to lifestyle intervention.

Link between organic nanovescicles from vegetable kingdom and human cell physiology: intracellular calcium signalling

BackgroundPlant-derived nanovesicles (PDNVs) are a novelty in medical and agrifood environments, with several studies exploring their functions and potential applications. Among fruits, apples (sp. Malus domestica) have great potential as PDNVs source, given their widespread consumption, substantial waste production, and recognized health benefits. Notably, apple-derived nanovesicles (ADNVs) can interact with human cell lines, triggering anti-inflammatory and antioxidant responses. This work is dedicated to the comprehensive biochemical characterization of apple-derived nanovesicles (ADNVs) through proteomic and lipidomic analysis, and small RNAs sequencing. This research also aims to shed light on the underlying mechanism of action (MOA) when ADNVs interface with human cells, through observation of intracellular calcium signalling in human fibroblasts, and to tackles differences in ADNVs content when isolated from fruits derived from integrated and organic production methods cultivars.ResultsThe ADNVs fraction is mainly composed of exocyst-positive organelles (EXPOs) and MVB-derived exosomes, identified through size and molecular markers (Exo70 and TET-3-like proteins). ADNVs’ protein cargo is heterogeneous and exhibits a diverse array of functions, especially in plant's protection (favouring ABA stress-induced signalling, pathogen resistance and Reactive Oxygen Species (ROS) metabolism). Noteworthy plant miRNAs also contribute to phytoprotection. In relation with human cells lines, ADNVs elicit spikes of intracellular Ca2+ levels, utilizing the cation as second messenger, and produce an antioxidant effect. Lastly, organic samples yield a substantial increase in ADNV production and are particularly enriched in bioactive lysophospholipids.ConclusionsWe have conclusively demonstrated that ADNVs confer an antioxidant effect upon human cells, through the initiation of a molecular pathway triggered by Ca2+ signalling. Within ADNVs, a plethora of bioactive proteins, small RNAs, and lipids have been identified, each possessing well-established functions within the realm of plant biology. While ADNVs predominantly function in plants, to safeguard against pathogenic agents and abiotic stressors, it is noteworthy that proteins with antioxidant power might act as antioxidants within human cells.Graphical

Functional characterization of SORL1 variants in cell-based assays to investigate variant pathogenicity.

SORLA, the protein encoded by the SORL1 gene, has an important role in recycling cargo proteins to the cell surface. While SORLA loss-of-function variants occur almost exclusively in Alzheimer's disease cases, the majority of SORL1 variants are missense variants that are individually rare and can have individual mechanisms how they impair SORLA function as well as have individual effect size on disease risk. However, since carriers mostly come from small pedigrees, it is challenging to determine variant penetrance, leaving clinical significance associated with most missense variants unclear. In this article, we present functional approaches to evaluate the pathogenicity of a SORL1 variant, p.D1105H. First, we generated our mutant receptor by inserting the D1105H variant into the full-length SORLA-WT receptor. Then using western blot analysis we quantified the effect of the mutation on maturation and shedding of the receptor for transfected cells, and finally applied a flow cytometry approach to quantify SORLA expression at the cell surface. The results showed decreased maturation, decreased shedding, and decreased cell surface expression of D1105H compared with wild-type SORLA. We propose how these approaches can be used to functionally assess the pathogenicity of SORL1 variants in the future. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Evoked Weibel-Palade Body Exocytosis Modifies the Endothelial Cell Surface by Releasing a Substrate-Selective Phosphodiesterase.

Weibel Palade bodies (WPB) are lysosome-related secretory organelles of endothelial cells. Commonly known for their main cargo, the platelet and leukocyte receptors von-Willebrand factor (VWF) and P-selectin, WPB play a crucial role in hemostasis and inflammation. Here, the authors identify the glycerophosphodiester phosphodiesterase domain-containing protein 5 (GDPD5) as a WPB cargo protein and show that GDPD5 is transported to WPB following uptake from the plasma membrane via an unique endocytic transport route. GDPD5 cleaves GPI-anchored, plasma membrane-resident proteins within their GPI-motif, thereby regulating their local activity. The authors identify a novel target of GDPD5 , the complement regulator CD59, and show that it is released from the endothelial surface by GDPD5 following WPB exocytosis. This results in increased deposition of complement components and can enhance local inflammatory and thrombogenic responses. Thus, stimulus-induced WPB exocytosis can modify the endothelial cell surface by GDPD5-mediated selective release of a subset of GPI-anchored proteins.

Proteolytic Vesicles Derived from Salmonella enterica Serovar Typhimurium-Infected Macrophages: Enhancing MMP-9-Mediated Invasion and EV Accumulation.

Proteolysis of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) plays a crucial role in the immune response to bacterial infections. Here we report the secretion of MMPs associated with proteolytic extracellular vesicles (EVs) released by macrophages in response to Salmonella enterica serovar Typhimurium infection. Specifically, we used global proteomics, in vitro, and in vivo approaches to investigate the composition and function of these proteolytic EVs. Using a model of S. Typhimurium infection in murine macrophages, we isolated and characterized a population of small EVs. Bulk proteomics analysis revealed significant changes in protein cargo of naïve and S. Typhimurium-infected macrophage-derived EVs, including the upregulation of MMP-9. The increased levels of MMP-9 observed in immune cells exposed to S. Typhimurium were found to be regulated by the toll-like receptor 4 (TLR-4)-mediated response to bacterial lipopolysaccharide. Macrophage-derived EV-associated MMP-9 enhanced the macrophage invasion through Matrigel as selective inhibition of MMP-9 reduced macrophage invasion. Systemic administration of fluorescently labeled EVs into immunocompromised mice demonstrated that EV-associated MMP activity facilitated increased accumulation of EVs in spleen and liver tissues. This study suggests that macrophages secrete proteolytic EVs to enhance invasion and ECM remodeling during bacterial infections, shedding light on an essential aspect of the immune response.

Emergent Spatiotemporal Organization in Stochastic Intracellular Transport Dynamics.

The interior of a living cell is an active, fluctuating, and crowded environment, yet it maintains a high level of coherent organization. This dichotomy is readily apparent in the intracellular transport system of the cell. Membrane-bound compartments called endosomes play a key role in carrying cargo, in conjunction with myriad components including cargo adaptor proteins, membrane sculptors, motor proteins, and the cytoskeleton. These components coordinate to effectively navigate the crowded cell interior and transport cargo to specific intracellular locations, even though the underlying protein interactions and enzymatic reactions exhibit stochastic behavior. A major challenge is to measure, analyze, and understand how, despite the inherent stochasticity of the constituent processes, the collective outcomes show an emergent spatiotemporal order that is precise and robust. This review focuses on this intriguing dichotomy, providing insights into the known mechanisms of noise suppression and noise utilization in intracellular transport processes, and also identifies opportunities for future inquiry.

Mesenchymal stem cell-derived extracellular vesicles exert pro-angiogenic and pro-lymphangiogenic effects in ischemic tissues by transferring various microRNAs and proteins including ITGa5 and NRP1

Mesenchymal stem cells/stromal cells (MSCs)-derived extracellular vesicles (EVs) mediate pro-regenerative effects in damaged ischemic tissues by regulating angiogenesis. MSCs-EVs modulate functions of cells including endogenous mature cells, progenitors and stem cells, resulting in restoration of blood flow. However, the mechanisms underlying such MSC-EV activity still remain poorly understood. The present study analyzes biological effects of bone marrow (BM) MSC-EVs on endothelial cells (ECs) in ischemic tissues both in in vitro and in vivo conditions and elucidates the molecular mechanisms underlying the tissue repair. MSC-EVs were isolated from murine BM-derived MSCs and their morphological, antigenic and molecular composition regarding protein and microRNA levels were evaluated to examine their properties. Global proteomic analysis demonstrated the presence in MSC-EVs of proteins regulating pro-regenerative pathways, including integrin α5 (Itgα5) and neuropilin-1 (NRP1) involved in lymphangiogenesis. MSC-EVs were also enriched in microRNAs regulating angiogenesis, TGF-β signaling and processes guiding cellular adhesion and interactions with extracellular matrix. The functional effects of MSC-EVs on capillary ECs in vitro included the increase of capillary-like tube formation and cytoprotection under normal and inflammatory conditions by inhibiting apoptosis. Notably, MSC-EVs enhanced also capillary-like tube formation of lymphatic ECs, which may be regulated by Itgα5 and NRP1. Moreover, in a mouse model of critical hind limb ischemia, MSC-EVs increased the recovery of blood flow in ischemic muscle tissue, which was accompanied with increased vascular density in vivo. This pro-angiogenic effect was associated with an increase in nitric oxide (NO) production via endothelial NO-synthase activation in ischemic muscles. Interestingly, MSC-EVs enhanced lymphangiogenesis, which has never been reported before. The study provides evidence on pro-angiogenic and novel pro-lymphangiogenic role of MSC-EVs on ECs in ischemic tissue mediated by their protein and miRNA molecular cargos. The results highlight Itgα5 and NRP1 carried by MSC-EVs as potential therapeutic targets to boost lymphangiogenesis.Graphical

TMED2 promotes glioma tumorigenesis by being involved in EGFR recycling transport

Aberrant epidermal growth factor receptor (EGFR) signaling is the core signaling commonly activated in glioma. The transmembrane emp24 protein transport domain protein 2 (TMED2) interacts with cargo proteins involved in protein sorting and transport between endoplasmic reticulum (ER) and Golgi apparatus. In this study, we found the correlation between TMED2 with glioma progression and EGFR signaling through database analysis. Moreover, we demonstrated that TMED2 is essential for glioma cell proliferation, migration, and invasion at the cellular levels, as well as tumor formation in mouse models, underscoring its significance in the pathobiology of gliomas. Mechanistically, TMED2 was found to enhance EGFR-AKT signaling by facilitating EGFR recycling, thereby providing the initial evidence of TMED2's involvement in the membrane protein recycling process. In summary, our findings shed light on the roles and underlying mechanisms of TMED2 in the regulation of glioma tumorigenesis and EGFR signaling, suggesting that targeting TMED2 could emerge as a promising therapeutic strategy for gliomas and other tumors associated with aberrant EGFR signaling.