Non-classical Notch signaling by MDA-MB-231 breast cancer cell-derived small extracellular vesicles promotes malignancy in poorly invasive MCF-7 cells.

Aberrant Notch signaling is implicated in breast cancer progression, and recent studies have demonstrated links between the Notch pathway components Notch1 and Notch1 intracellular domain (N1ICD) with poor clinical outcomes. Growing evidence suggests that Notch signaling can be regulated by small extracellular vesicles (SEVs). Here, we used breast cancer cell models to examine whether SEVs are involved in functional Notch signaling. We found that Notch components are packaged into MDA-MB-231- and MCF-7-derived SEVs, although higher levels of N1ICD were detected in SEVs from the more aggressive MDA-MB-231 cell line than from poorly invasive MCF-7 cells. SEV-Notch components were functional, as SEVs cargo from MDA-MB-231 cells induced the expression of Notch target genes in MCF-7 cells and triggered a more invasive and proliferative phenotype concomitant with the acquisition of mesenchymal features. Neutralization of the N1ICD cargo in MDA-MB-231-derived SEVs significantly reduced their potential to enhance the aggressiveness of MCF-7 cells in vitro and in a xenograft model. Overall, our results indicate that a SEV-mediated non-classical pathway of Notch signal transduction in breast cancer models bypasses the need for classical ligand-receptor interactions, which may have important implications in cancer.

Polymer Conjugation of Docosahexaenoic Acid Potentiates Cardioprotective Therapy in Preclinical Models of Myocardial Ischemia/Reperfusion Injury.

While coronary angioplasty represents an effective treatment option following acute myocardial infarction, the reperfusion of the occluded coronary artery can prompt ischemia-reperfusion (I/R) injury that significantly impacts patient outcomes. As ω-3 polyunsaturated fatty acids (PUFAs) have proven, yet limited cardioprotective abilities, an optimized polymer-conjugation approach is reported that improves PUFAs bioavailability to enhance cardioprotection and recovery in animal models of I/R-induced injury. Poly-l-glutamic acid (PGA) conjugation improves the solubility and stability of di-docosahexaenoic acid (diDHA) under physiological conditions and protects rat neonatal ventricular myocytes from I/R injury by reducing apoptosis, attenuating autophagy, inhibiting reactive oxygen species generation, and restoring mitochondrial membrane potential. Enhanced protective abilities are associated with optimized diDHA loading and evidence is provided for the inherent cardioprotective potential of PGA itself. Pretreatment with PGA-diDHA before reperfusion in a small animal I/R model provides for cardioprotection and limits area at risk (AAR). Furthermore, the preliminary findings suggest that PGA-diDHA administration in a swine I/R model may provide cardioprotection, limit edema and decrease AAR. Overall, the evaluation of PGA-diDHA in relevant preclinical models provides evidence for the potential of polymer-conjugated PUFAs in the mitigation of I/R injury associated with coronary angioplasty.

Extracellular Vesicles Secreted by Hypoxic AC10 Cardiomyocytes Modulate Fibroblast Cell Motility.

Extracellular vesicles (EVs) are small membrane vesicles secreted by most cell types with important roles in cell-to-cell communication. To assess their relevance in the context of heart ischemia, EVs isolated from the AC10 ventricular cardiomyocyte cell line (CM-EVs), exposed to normoxia (Nx) or hypoxia (Hx), were incubated with fibroblasts (Fb) and endothelial cells (EC). CM-EVs were studied using electron microscopy, nanoparticle tracking analysis (NTA), western blotting and proteomic analysis. Results showed that EVs had a strong preference to be internalized by EC over fibroblasts, suggesting an active exosome-based communication mechanism between CM and EC in the heart. In Matrigel tube-formation assays, Hx CM-EVs were inferior to Nx CM-EVs in angiogenesis. By contrast, in a wound-healing assay, wound closure was faster in fibroblasts treated with Hx CM-EVs than with Nx CM-EVs, supporting a pro-fibrotic effect of Hx CM-EVs. Overall, these observations were consistent with the different protein cargoes detected by proteomic analysis under Nx and Hx conditions and the biological pathways identified. The paracrine crosstalk between CM-EVs, Fb, and EC in different physiological conditions could account for the contribution of CM-EVs to cardiac remodeling after an ischemic insult.

Analysis of Exosome Transfer in Mammalian Cells by Fluorescence Recovery after Photobleaching.

During the course of evolution, prokaryote and eukaryote cells have developed elegant and to some extent analogous strategies to communicate with each other and to adapt to their surrounding environment. Eukaryotic cells communicate with each other through direct interaction via juxtracrine signaling and/or by secreting soluble factors. These secreted factors can subsequently act on the cell itself (autocrine signaling) or interact with neighboring (paracrine signaling) and distant (endocrine signaling) cells. The transmission of signals between cells and tissues has been traditionally thought to be regulated by a protein-based signaling system. Typically, proteins destined for secretion into the extracellular milieu by exocytosis contain a canonical secretion-targeting sequence ( Théry et al., 2002 ). However, proteins with a non-continuous and stimulus-dependent secretion, proteins that do not contain a canonical secretion-targeting sequence, and species that might be too labile within the extracellular environment (DNA, mRNA, peptides, metabolites, miRNA and other RNA species), can be secreted in small membranous extracellular vesicles (EVs) in a specific manner ( Hagiwara et al., 2014 ). Exosomes represent one broad class of these secreted membrane vesicles with a diameter of 30-130 nm ( Cocucci et al., 2009 ; Théry et al., 2009 ; Kowal et al., 2014 ), which are formed inside the secreting cells in endosomal compartments called multivesicular bodies. Molecules loaded into exosomes as well as the intensity of the exosome transfer between cells are important parameters for the subsequent conditioning of recipient cells. Current knowledge on secretion of exosomes and their internalization in recipient cells remains incomplete. It is known that secretion intensity of exosomes varies according to the cellular type and its physiological state ( Garcia et al., 2016 ). Moreover, the different combination of transmembrane proteins on the surface of exosomes that facilitate the adhesion to the cell-extracellular matrix vary the avidity with which a recipient cell captures exosomes ( Hoshino et al., 2015 ). Here, we have developed an in vitro system by which the transfer of exosomes between cells in co-culture can be quantified using FRAP ('Fluorescence Recovery After Photobleaching') technology. This protocol has been used to analyze the effects of exosome transfer of hypoxia inducible factor 1-α (HIF-1α) in Mesenchymal Stem Cells (MSC; HIF-MSC) to Human Umbilical Cord Vein Endothelial Cells (HUVEC) (Gonzalez-King et al., 2017).

Mesenchymal Stem Cell Migration and Proliferation Are Mediated by Hypoxia-Inducible Factor-1α Upstream of Notch and SUMO Pathways.

Mesenchymal stem cells (MSCs) are effective in treating several pathologies. We and others have demonstrated that hypoxia or hypoxia-inducible factor 1 alpha (HIF-1α) stabilization improves several MSC functions, including cell adhesion, migration, and proliferation, thereby increasing their therapeutic potential. To further explore the mechanisms induced by HIF-1α in MSCs, we studied its relationship with Notch signaling and observed that overexpression of HIF-1α in MSCs increased protein levels of the Notch ligands Jagged 1-2 and Delta-like (Dll)1, Dll3, and Dll4 and potentiated Notch signaling only when this pathway was activated. Crosstalk between HIF and Notch resulted in Notch-dependent migration and spreading of MSCs, which was abolished by γ-secretase inhibition. However, the HIF-1-induced increase in MSC proliferation was independent of Notch signaling. The ubiquitin family member, small ubiquitin-like modifier (SUMO), has important functions in many cellular processes and increased SUMO1 protein levels have been reported in hypoxia. To investigate the potential involvement of SUMOylation in HIF/Notch crosstalk, we measured general SUMOylation levels and observed increased SUMOylation in HIF-1-expressing MSCs. Moreover, proliferation and migration of MSCs were reduced in the presence of a SUMOylation inhibitor, and this effect was particularly robust in HIF-MSCs. Immunoprecipitation studies demonstrated SUMOylation of the intracellular domain of Notch1 (N1ICD) in HIF-1-expressing MSCs, which contributed to Notch pathway activation and resulted in increased levels of N1ICD nuclear translocation as assessed by subcellular fractionation. SUMOylation of N1ICD was also observed in HEK293T cells with stabilized HIF-1α expression, suggesting that this is a common mechanism in eukaryotic cells. In summary, we describe, for the first time, SUMOylation of N1ICD, which is potentiated by HIF signaling. These phenomena could be relevant for the therapeutic effects of MSCs in hypoxia or under conditions of HIF stabilization.

Hypoxia Inducible Factor-1α Potentiates Jagged 1-Mediated Angiogenesis by Mesenchymal Stem Cell-Derived Exosomes.

Insufficient vessel growth associated with ischemia remains an unresolved issue in vascular medicine. Mesenchymal stem cells (MSCs) have been shown to promote angiogenesis via a mechanism that is potentiated by hypoxia. Overexpression of hypoxia inducible factor (HIF)-1α in MSCs improves their therapeutic potential by inducing angiogenesis in transplanted tissues. Here, we studied the contribution of exosomes released by HIF-1α-overexpressing donor MSCs (HIF-MSC) to angiogenesis by endothelial cells. Exosome secretion was enhanced in HIF-MSC. Omics analysis of miRNAs and proteins incorporated into exosomes pointed to the Notch pathway as a candidate mediator of exosome communication. Interestingly, we found that Jagged1 was the sole Notch ligand packaged into MSC exosomes and was more abundant in HIF-MSC than in MSC controls. The addition of Jagged1-containing exosomes from MSC and HIF-MSC cultures to endothelial cells triggered transcriptional changes in Notch target genes and induced angiogenesis in an in vitro model of capillary-like tube formation, and both processes were stimulated by HIF-1α. Finally, subcutaneous injection of Jagged 1-containing exosomes from MSC and HIF-MSC cultures in the Matrigel plug assay induced angiogenesis in vivo, which was more robust when they were derived from HIF-MSC cultures. All Jagged1-mediated effects could be blocked by prior incubation of exosomes with an anti-Jagged 1 antibody. All together, the results indicate that exosomes derived from MSCs stably overexpressing HIF-1α have an increased angiogenic capacity in part via an increase in the packaging of Jagged1, which could have potential applications for the treatment of ischemia-related disease. Stem Cells 2017;35:1747-1759.

Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes.

AIMS: Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship, which is essential for maintaining the metabolic requirements of the heart. Little is known about the mechanisms that regulate nutrient flow from ECs to associated CMs, especially in situations of acute stress when local active processes are required to regulate endothelial transport. We examined whether CM-derived exosomes can modulate glucose transport and metabolism in ECs. METHODS AND RESULTS: In conditions of glucose deprivation, CMs increase the synthesis and secretion of exosomes. These exosomes are loaded with functional glucose transporters and glycolytic enzymes, which are internalized by ECs, leading to increased glucose uptake, glycolytic activity, and pyruvate production in recipient cells. CONCLUSION: These findings establish CM-derived exosomes as key components of the cardio-endothelial communication system which, through intercellular protein complementation, would allow a rapid response from ECs to increase glucose transport and a putative uptake of metabolic fuels from blood to CMs. This CM-EC protein complementation process might have implications for metabolic regulation in health and disease.

Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells.

Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship that is essential for maintaining normal development and function in the heart. Little is known about the mechanisms that regulate cardiac and endothelial crosstalk, particularly in situations of acute stress when local active processes are required to regulate endothelial function. We examined whether CM-derived exosomes could modulate endothelial function. Under conditions of glucose deprivation, immortalized H9C2 cardiomyocytes increase their secretion of exosomes. CM-derived exosomes are loaded with a broad repertoire of miRNA and proteins in a glucose availability-dependent manner. Gene Ontology (GO) analysis of exosome cargo molecules identified an enrichment of biological process that could alter EC activity. We observed that addition of CM-derived exosomes to ECs induced changes in transcriptional activity of pro-angiogenic genes. Finally, we demonstrated that incubation of H9C2-derived exosomes with ECs induced proliferation and angiogenesis in the latter. Thus, exosome-mediated communication between CM and EC establishes a functional relationship that could have potential implications for the induction of local neovascularization during acute situations such as cardiac injury.