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

The effect of earned versus house money on price bubble formation in experimental asset markets

Does house money exacerbate price bubbles? We compare house money asset market experiments with an earned money treatment where initial portfolios are constructed from a real effort task. Bubbles occur; however, trading volumes and earnings dispersion are significantly higher with house money. We investigate the role of cognitive ability in accounting for the differences in earnings distribution across treatments by using the cognitive reflection test (CRT). Low CRT subjects earned less than high CRT subjects. Low CRT subjects were net purchasers (sellers) of shares when the price was above (below) fundamental value. The opposite was true for high CRT subjects

Head-to-head Comparison of Relevant Cell Sources of Small Extracellular Vesicles for Cardiac Repair: Superiority of Embryonic Stem Cells

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI

Estudio de las Implicaciones de la Via de Señalización de Notch en Cardioregeneración y Cáncer

[ES] Las células de los mamíferos secretan una gran variedad de vesículas extracelulares (EV) que participan activamente en la comunicación celular. Entre ellas, los exosomas son un tipo de EV cuyo tamaño está comprendido entre los 20 y 150 nanómetros que juegan un papel importante en la comunicación celular. De este modo, se ha descrito el papel de los exosomas en distintos procesos como la regeneración de tejidos, la respuesta inmunitaria o en la progresión de diferentes tipos de cánceres. Por otro lado, se ha observado que la actividad de la vía de señalización de Notch juega un papel importante en procesos de diferenciación celular, la angiogénesis o la proliferación, entre otros. Además, se ha relacionado con la aparición y progresión de un gran número de patologías, como el cáncer. Trabajos recientes han advertido que la actividad de la vía de Notch puede regularse mediante un nuevo mecanismo de señalización mediado por exosomas. En este trabajo se profundizó en este nuevo mecanismo de señalización de la vía de Notch en dos contextos diferentes como son las terapias regenerativas y el cáncer. Un trabajo previo de nuestro laboratorio demostró que las células madre mesenquimales (MSC) que mantenían una expresión aumentada del factor inducible por hipoxia 1α (HIF-1α; HIF-MSC) aumentaban el potencial terapéutico de las MSC nativas en un modelo de infarto agudo de miocardio (IAM) en rata a través de un aumento de la angiogénesis y una reducción de la fibrosis. Varios trabajos han estudiado la relación entre la vía de señalización de Notch y HIF-1α; así como la influencia de ambos en el proceso angiogénico. Por estos motivos, en este trabajo nos interesamos por evaluar la relación de ambas vías y su efecto sobre la angiogénesis a través de los exosomas liberados por las HIF-MSC y las MSC. Los resultados permitieron observar que la expresión de HIF-1α indujo un aumento en la secreción y la transferencia de exosomas de MSC a cultivos primarios de células endoteliales. Además, se observó que los exosomas derivados de HIF-MSC tenían un mayor potencial angiogénico tanto in vitro como in vivo en parte debido a una mayor incorporación del ligando de la vía de Notch Jagged1. Se ha observado que los exosomas derivados de células tumorales contribuyen a la progresión tumoral a través de mecanismos como la activación de la transición epitelio mesénquima (EMT), el establecimiento de nichos pre-metastáticos o la inmunomodulación. Así, se sabe que la vía de señalización de Notch es capaz de activar la EMT durante la progresión de distintos tipos de cánceres. Así mismo, los cánceres de mama de peor prognosis han sido relacionados con una desregulación de la vía de señalización de Notch. Por estas razones, nos inquietó la posibilidad de que la señalización de la vía de Notch a través de exosomas estuviera influyendo en la progresión del cáncer de mama. De este modo, estudiamos la presencia de componentes de la vía de señalización de Notch en los exosomas procedentes de dos líneas tumorales de cáncer de mama, una más agresiva (MDA-MB-231) y otra menos agresiva (MCF-7). Así, se observó que varios componentes de Notch se sobreexpresaron en los exosomas procedentes de MDA-MB-231 en comparación con los procedentes de MCF-7. Fue de particular interés la detección del dominio intracelular con actividad transcripcional de Notch1 (N1ICD) sobreexpresado en los exosomas procedentes de MDA-MB-231. Debido a que previamente había sido demostrado que una sobreexpresión de N1ICD en MCF-7 aumentaba su tumorigenicidad a través de una inducción de la EMT, se evaluó el efecto de los componentes de Notch incorporados en los exosomas procedentes de ambas líneas tumorales sobre cultivos de MCF-7. Nuestros resultados sugieren que los componentes de Notch incorporados en los exosomas procedentes de MDA-MB-231 son funcionales y contribuyen a la induc[CA] Les cèl·lules dels mamífers secreten una gran varietat de vesícules extracel·lulars (EV) que participen activament en la comunicació celular. Entre elles, els exosomes són un tipus de EV amb una grandària compresa entre els 20 i 150 nanòmetres que juguen un paper important en la comunicació celular. D'aquesta manera, s'ha descrit el paper dels exosomes en diferents processos com la regeneració de teixits, la resposta immunitària o en la progressió de diferents tipus de càncers. D'altra banda, s'ha observat que l'activitat de la via de senyalització de Notch juga un paper molt important en processos de diferenciació cel·lular, la angiogénesis o la proliferació, entre altres. A més, s'ha relacionat amb l'aparició i progressió d'un gran nombre de patologies, com el càncer. Treballs recents han advertit que l'activitat de la via de Notch pot regular-se mitjançant un nou mecanisme de senyalització mediat per exosomes. En aquest treball es va aprofundir en aquest nou de mecanisme de senyalització de la via de Notch en dos contextos diferents com són les teràpies regeneratives i el càncer. Un treball publicat pel nostre equip de treball va demostrar que les cèl·lules mare mesenquimals (MSC) que mantenien una expressió augmentada del factor inducible per hipòxia 1α (HIF-1α; HIF-MSC) augmentaven el potencial terapèutic de les MSC natives en un model d'infart agut de miocardi (IAM) en rata a través d'un augment de la angiogénesis i una reducció de la fibrosi. Diversos treballs han estudiat la relació entre la via de senyalització de Notch i HIF-1α; així com la influència de tots dos en el procés angiogénic. Per aquests motius, en aquest treball ens vam interessar en estudiar la relació de totes dues vies i el seu efecte sobre la angiogénesis a través dels exosomes alliberats per les HIF-MSC i les MSC. Els resultats van permetre observar que l'expressió de HIF-1α va induir un augment en la secreció i la transferència d'exosomes de MSC a cultius primaris de cèl·lules endotelials. A més, es va observar que els exosomes derivats de HIF-MSC tenien un major potencial angiogénic tant in vitro com in vivo en part a causa d'una major incorporació del lligant de la via de Notch Jagged1. S'ha observat que els exosomes procedents de cèl·lules tumorals contribueixen a la progressió tumoral a través de mecanismes com l'activació de la transició epiteli mesènquima (EMT), l'establiment de nínxols pre-metastàtics o la inmunomodulació. En aquest sentit, se sap que la via de senyalització de Notch és capaç d'activar l'EMT durant la progressió de diferents tipus de càncers. Així mateix, els càncers de mama de pitjor prognosi han sigut reiteradament relacionats amb una desregulació de la via de senyalització de Notch. Per açò, ens va inquietar la possibilitat que la senyalització de la via de Notch a través d'exosomes estiguera influint en la progressió del càncer de mama. D'aquesta manera, estudiem la presència de components de la via de senyalització de Notch en els exosomes procedents de dues línies tumorals de càncer de mama, una més agressiva (MDA-MB-231) i una altra menys agressiva (MCF-7). Així, es va observar que diversos components de Notch es sobreexpresaben en els exosomes procedents de MDA-MB-231 en comparació amb els procedents de MCF-7. Va ser de particular interés la detecció del domini intracel·lular amb activitat transcripcional de Notch1 (N1ICD) sobreexpresat en els exosomes procedents de MDA-MB-231. Debut a que havia sigut demostrat que una sobreexpressió de N1ICD en MCF-7 augmentava la seua tumorigenicidad a través d'una inducció de l'EMT, es va evaluar l'efecte dels components de Notch incorporats en els exosomes procedents de les dues línies tumorals sobre cultius de MCF-7. Els nostres resultats suggereixen que els components de Notch incorporats en els exosomes procedents de MDA-MB-231 eren funcionals i contribuïen a la[EN] Mammal cells secrete a great variety of extracellular vesicles (EV) that actively participate in intercellular communication. Among them, exosomes are a type of EV whose size is between 20 and 150 nanometers and play an important role in intercellular communication. In this way, the role of exosomes in different processes such as tissue regeneration, the immune response or in the progression of different types of cancers has been described. On the other hand, it has been observed that the Notch signaling pathway plays a very important role in processes such as cell differentiation, angiogenesis or proliferation, among others. In addition, it has been related to the appearance and progression of a large number of pathologies, such as cancer. Recent works have shown that the activity of the Notch pathway can be regulated by a new signaling mechanism mediated by exosomes. In this work, we deepened this new signaling mechanism of the Notch signaling pathway in two different contexts: the regenerative therapies and cancer. A work previously published by our laboratory showed that mesenchymal stem cells (MSC) that maintained an increased expression of hypoxia-inducible factor 1α (HIF-1α; HIF-MSC) increased the therapeutic potential of native MSC in a model of acute myocardial infarction (AMI) in rats through an increase in angiogenesis and a reduction in fibrosis. Several studies have studied the relationship between the Notch signaling pathway and HIF-1α as well as the influence of both in the angiogenic process. For these reasons, in this work we were interested in evaluating the relationship of both pathways and their effect on angiogenesis through exosomes released by HIF-MSCs and MSCs. The results showed that the expression of HIF-1α induced an increase in the secretion and the transfer of exosomes of MSC to primary cultures of endothelial cells. In addition, it was observed that exosomes derived from HIF-MSC had a greater angiogenic potential both in vitro and in vivo in part due to a greater incorporation of the Notch ligand Jagged1. In the last decade, it has been observed that exosomes from tumor cells contribute to tumor progression through mechanisms such as the activation of epithelial to mesenchymal transition (EMT), the establishment of pre-metastatic niches orimmunomodulation. In this sense, it is known that the Notch signaling pathway is capable of activating EMT during the progression of different types of cancers. Likewise, breast cancers of bad prognosis have been repeatedly related to a deregulation of the Notch signaling pathway. For these reasons, we were concerned about the possibility that the signaling of the Notch pathway through exosomes was influencing the progression of breast cancers. In this way, we studied the presence of Notch signaling pathway components in exosomes from the metastatic and more aggressive MDA-MB-231 and the non-metastatic and less aggressive MCF-7 cell lines. It was observed that several Notch components were overexpressed in exosomes from MDA-MB-231 compared to those from MCF-7. Of particular interest was the detection of the intracellular domain with transcriptional activity of Notch1 (N1ICD) overexpressed in MDA-MB-231 exosomes. It has been previously demonstrated that an overexpression of N1ICD in MCF-7 increases its tumorigenicity through an induction of EMT. Because of this, we evaluated the effect of Notch components incorporated in exosomes from both tumoral cell lines on MCF-7 cultures. Our results suggested that Notch components incorporated into MDA-MB-231 exosomes were functional and contributed to the induction of EMT when added to MCF-7 cultures in part due to N1ICD activity.González-King Garibotti, H. (2019). Estudio de las Implicaciones de la Via de Señalización de Notch en Cardioregeneración y Cáncer [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124343TESI

Aproximación a las implicaciones terapéuticas en el infarto agudo de miocardio de exosomas extraídos de células MSC y MSC-HIF-1α

[EN] Acute Myocardial Infarction (AMI) still is an unsolved issue nowadays. That’s why, the good results obtained over the last years in the treatment of AMI with mesenquimal stem cells (MSC) make them a good alternative therapie instead of conventional methods. Previous reports demonstrated that hypoxia improves MSC self-renewal and therapeutic effect. Considering that hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the adaptative response to hypoxia, as stated in a work published by our work team, showed that overexpression of HIF-1α in this cells (MSC-HIF) could be a good strategy to achieve better therapeutics in this cells. In the first approach, the general thought was that MSC and MSC-HIF-1α were acting through transdifferentiation into other cell types such, for example, endothelial cells or myocites. However, this wasn’t taking place, that’s why started to gain importance that the beneficial effects of mesenchymal stem cells were due to the release of soluble factors. It is longly known that the Notch pathway is involved in many important processes, such embryo development, apoptosis, tumor growth, differentiation or angiogenesis among others. In this work we have studied the effect exerted by exosomes released by MSC transfected with HIF-1α or not in endothelial cell biology. The results show that exosomes, released by MSC-HIF and MSC are loaded with the ligand Jagged-1, one of the activating ligands of Notch pathway and that these exosomes are capable of activating this pathway in endothelial cells. Many others studies report that this kind of activation results in enhanced proliferation and vessel formation capacity, which could have important therapeutic implications and partly explain the paracrine effect exerted by the MSC on various cell types.[ES] Hoy en día el infarto agudo de miocardio (IAM) sigue sin tener una solución efectiva. De este modo, los buenos resultados obtenidos en los últimos años en el tratamiento del IAM mediante terapia celular con células madre adultas mesenquimales (MSC) hace que se postulen como una posible terapia alternativa a los métodos convencionales. Se ha visto que el precondicionamiento a un ambiente de hipoxia mejora el potencial terapéutico de las MSC, por esta razón, un trabajo publicado por nuestro equipo de trabajo utilizó MSC que sobreexpresaban el factor de respuesta a hipoxia 1α (HIF-1α; MSC-HIF) que juega un papel primordial en la adaptación a la hipoxia. En un primer acercamiento se pensó que el efecto beneficioso de las MSC en la terapia celular venía estrechamente ligado a la capacidad de transdiferenciación de dichas células a otros tipos celulares como endotelio o miocardio, no obstante, no se corroboraba con lo que se observó a posteriori. Es por eso que adquirió mayor importancia la hipótesis paracrina del efecto terapéutico de las MSC y MSC-HIF. Se sabe desde hace mucho tiempo que la vía de Notch es una vía implicada en un gran número de procesos importantes, tales como el desarrollo embriogénico, la apoptosis, el desarrollo de tumores, la diferenciación o la angiogénesis entre otros. En el presente trabajo vemos el efecto que ejercen los exosomas liberados por células MSC transfectadas o no con HIF-1α en la biología de las células endoteliales. Los resultados muestran que los exosomas liberados por MSC y MSC-HIF van cargados con el ligando Jagged-1 de la vía de Notch y que son capaces de activar dicha vía en células endoteliales. Muchos estudios han demostrado que este tipo de señalización se traduce en una mayor proliferación y capacidad de formar vasos, lo que podría tener importantes implicaciones terapéuticas y explicar en parte el efecto paracrino ejercido por las MSC sobre diversos tipos celulares. (español)González-King Garibotti, H. (2015). Aproximación a las implicaciones terapéuticas en el infarto agudo de miocardio de exosomas extraídos de células MSC y MSC-HIF-1α. http://hdl.handle.net/10251/64834Archivo delegad

Oncostatin M-Enriched Small Extracellular Vesicles Derived from Mesenchymal Stem Cells Prevent Isoproterenol-Induced Fibrosis and Enhance Angiogenesis

Myocardial fibrosis is a pathological hallmark of cardiac dysfunction. Oncostatin M (OSM) is a pleiotropic cytokine that can promote fibrosis in different organs after sustained exposure. However, OSM released by macrophages during cardiac fibrosis suppresses cardiac fibroblast activation by modulating transforming growth factor beta 1 (TGF-β1) expression and extracellular matrix deposition. Small extracellular vesicles (SEVs) from mesenchymal stromal cells (MSCs) are being investigated to treat myocardial infarction, using different strategies to bolster their therapeutic ability. Here, we generated TERT-immortalized human MSC cell lines (MSC-T) engineered to overexpress two forms of cleavage-resistant OSM fused to CD81TM (OSM-SEVs), which allows the display of the cytokine at the surface of secreted SEVs. The therapeutic potential of OSM-SEVs was assessed in vitro using human cardiac ventricular fibroblasts (HCF-Vs) activated by TGF-β1. Compared with control SEVs, OSM-loaded SEVs reduced proliferation in HCF-V and blunted telo-collagen expression. When injected intraperitoneally into mice treated with isoproterenol, OSM-loaded SEVs reduced fibrosis, prevented cardiac hypertrophy, and increased angiogenesis. Overall, we demonstrate that the enrichment of functional OSM on the surface of MSC-T-SEVs increases their potency in terms of anti-fibrotic and pro-angiogenic properties, which opens new perspectives for this novel biological product in cell-free-based therapies

miR-4732-3p in Extracellular Vesicles From Mesenchymal Stromal Cells Is Cardioprotective During Myocardial Ischemia

[EN] Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are an emerging alternative to cell-based therapies to treat many diseases. However, the complexity of producing homogeneous populations of EVs in sufficient amount hampers their clinical use. To address these limitations, we immortalized dental pulp-derived MSC using a human telomerase lentiviral vector and investigated the cardioprotective potential of a hypoxia-regulated EV-derived cargo microRNA, miR-4732-3p. We tested the compared the capacity of a synthetic miR-4732-3p mimic with EVs to confer protection to cardiomyocytes, fibroblasts and endothelial cells against oxygen-glucose deprivation (OGD). Results showed that OGD-induced cardiomyocytes treated with either EVs or miR-4732-3p showed prolonged spontaneous beating, lowered ROS levels, and less apoptosis. Transfection of the miR-4732-3p mimic was more effective than EVs in stimulating angiogenesis in vitro and in vivo and in reducing fibroblast differentiation upon transforming growth factor beta treatment. Finally, the miR-4732-3p mimic reduced scar tissue and preserved cardiac function when transplanted intramyocardially in infarcted nude rats. Overall, these results indicate that miR-4732-3p is regulated by hypoxia and exerts cardioprotective actions against ischemic insult, with potential application in cell-free-based therapeutic strategies.This work was supported in part by grants from the Instituto de Salud Carlos III PI19/0245, RD16/0011/0004, cofunded by FEDER Una manera de hacer Europa and INNCON-2020-6-CARVEMO from Agencia Valenciana de la Innovación. This work was also supported by predoctoral fellowships to RS-S, MG-F, IR, and MB grants ACIF/2017/318, ACIF/2018/254, ACIF2019/257, and ACIF2019/250 from the Conselleria de Sanitat Universal i Salut Pública and co-financed by the European Union through the Operational Programme European Regional Development Fund (FEDER) of the Valencian Community 2014 2020.Sánchez-Sánchez, R.; Gómez-Ferrer, M.; Reinal-Ferre, I.; Buigues, M.; Villanueva-Bádenas, E.; Ontoria-Oviedo, I.; Hernándiz, A.... (2021). miR-4732-3p in Extracellular Vesicles From Mesenchymal Stromal Cells Is Cardioprotective During Myocardial Ischemia. Frontiers in Cell and Developmental Biology. 9. https://doi.org/10.3389/fcell.2021.734143

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

<div><p>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.</p></div

H9C2-derived U exosomes modify gene transcription in HUVEC.

<p>(A) qPCR analysis of angiogenesis related genes from HUVEC incubated for 40 min with 20 μg/ml of U exosomes derived from H9C2 with or without glucose starvation (+/- St) for 48 h. Results are presented normalized to the control treatment (PBS).</p

KEGG pathways associated with miRNAs over-expressed in +St conditions.

<p>KEGG pathways associated with miRNAs over-expressed in +St conditions.</p