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.

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.

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. 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. 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.

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.

Regulation of circulating free fatty acid (FFA) levels and delivery is crucial to maintain tissue homeostasis. Exosomes are nanomembranous vesicles that are released from diverse cell types and mediate intercellular communication by delivering bioactive molecules. Here, we sought to investigate the uptake of FFAs by circulating exosomes, the delivery of FFA-loaded exosomes to cardiac cells and the possible role of the FFA transporter CD36 in these processes. Circulating exosomes were purified from the serum of healthy donors after an overnight fast (F) or 20 minutes after a high caloric breakfast (postprandial, PP). Western blotting, Immunogold Electron Microscopy and FACS analysis of circulating exosomes showed that CD36 was expressed under both states, but was higher in postprandial-derived exosomes. Flow cytometry analysis showed that circulating exosomes were able to take-up FFA directly from serum. Importantly, preincubation of exosomes with a blocking CD36 antibody significantly impeded uptake of the FFA analogue BODIPY, pointing to the role of CD36 in FFA exosomal uptake. Finally, we found that circulating exosomes could delivery FFA analogue BODIPY into cardiac cells ex vivo and in vivo in a mice model. Overall, our results suggest a novel mechanism in which circulating exosomes can delivery FFAs from the bloodstream to cardiac tissue. Further studies will be necessary to understand this mechanism and, in particular, its potential involvement in metabolic pathologies such as obesity, diabetes and atherosclerosis.

Arrhythmogenic cardiomyopathy (ACM) is a disorder characterized by a progressive ventricular myocardial replacement by fat and fibrosis which lead to ventricular arrhythmias and sudden cardiac death. Mutations in the desmosomal gene Plakophilin-2 (PKP2) accounts for >40% of all known mutations, generally causing a truncated protein. In a PKP2 truncated mouse model, we hypothesize that content of transgene, endurance training and aging will be determinant in disease progression. In addition, we investigated the molecular defects associated with the phenotype in this model. We developed a transgenic mouse model containing a truncated PKP2 (PKP2-Ser329) and generated three transgenic lines expressing increasing transgene content. The pathophysiological features of ACM in this model were assessed. While we did not observe fibro-fatty replacement, ultrastructural defects were exhibited. Moreover, we observed transgene content-dependent development of structural (ventricle dilatation and dysfunction) and electrophysiological anomalies in mice (PR interval and QRS prolongation and arrhythmia induction). In concordance with pathological defects, we detected a content reduction and remodeling of the structural proteins Desmocollin-2, Plakoglobin, native Plakophilin-2, Desmin and β-Catenin as well as the electrical coupling proteins Connexin 43 and cardiac sodium channel (Na_v1.5). Surprisingly, we observed structural but not electrophysiological abnormalities only in trained and old mice. We demonstrated that truncated PKP2 provokes ACM in absence of fibro-fatty replacement in the mouse. Transgene dose is essential to reveal the pathology, whereas aging and endurance training trigger limited phenotype. Molecular abnormalities underlay the structural and electrophysiological defects.