Extracellular vesicles (EVs) include a variety of nanosized vesicles released to the extracellular microenvironment by the vast majority of cells transferring bioactive lipids, proteins, mRNA, miRNA ...or non-coding RNA, as means of intercellular communication. Remarkably, among other fields of research, their use has become promising for immunomodulation, tissue repair and as source for novel disease-specific molecular signatures or biomarkers. However, a major challenge is to define accurate, reliable and easily implemented techniques for EV isolation due to their nanoscale size and high heterogeneity. In this context, differential ultracentrifugation (dUC) has been the most widely used laboratory methodology, but alternative procedures have emerged to allow purer EV preparations with easy implementation. Here, we present and discuss the most used of the different EV isolation methods, focusing on the increasing impact of size exclusion chromatography (SEC) on the resulting EV preparations from in vitro cultured cells-conditioned medium and biological fluids. Comparatively, low protein content and cryo-electron microscopy analysis show that SEC removes most of the overabundant soluble plasma proteins, which are not discarded using dUC or precipitating agents, while being more user friendly and less time-consuming than gradient-based EV isolation. Also, SEC highly maintains the major EVs’ characteristics, including vesicular structure and content, which guarantee forthcoming applications. In sum, together with scaling-up possibilities to increase EV recovery and manufacturing following high-quality standards, SEC could be easily adapted to most laboratories to assist EV-associated biomarker discovery and to deliver innovative cell-free immunomodulatory and pro-regenerative therapies.
Left ventricular (LV) remodeling after myocardial infarction (MI) is promoted by an intense fibrotic response, which could be targeted by the anti-fibrotic drug pirfenidone. We explored the ...relationship between protein modulation by pirfenidone and post-MI remodeling, based on molecular information and transcriptomic data from a swine model of MI. We identified 6 causative motives of post-MI remodeling (cardiomyocyte cell death, impaired myocyte contractility, extracellular matrix remodeling and fibrosis, hypertrophy, renin-angiotensin-aldosterone system activation, and inflammation), 4 pirfenidone targets and 21 bioflags (indirect effectors). Pirfenidone had a more widespread action than gold-standard drugs, encompassing all 6 motives, with prominent effects on p38γ-MAPK12, the TGFβ1-SMAD2/3 pathway and other effector proteins such as matrix metalloproteases 2 and 14, PDGFA/B, and IGF1. A bioinformatic approach allowed to identify several possible mechanisms of action of pirfenidone with beneficial effects in the post-MI LV remodeling, and suggests additional effects over guideline-recommended therapies.
Cardiac fibrosis is characterized by the deposition of extracellular matrix proteins in the spaces between cardiomyocytes following both acute and chronic tissue damage events, resulting in the ...remodeling and stiffening of heart tissue. Fibrosis plays an important role in the pathogenesis of many cardiovascular disorders, including heart failure and myocardial infarction. Several studies have identified fibroblasts, which are induced to differentiate into myofibroblasts in response to various types of damage, as the most important cell types involved in the fibrotic process. Some drugs, such as inhibitors of the renin–angiotensin–aldosterone system, have been shown to be effective in reducing cardiac fibrosis. There are currently no drugs with primarily anti-fibrotic action approved for clinical use, as well as the evidence of a clinical efficacy of these drugs is extremely limited, despite the numerous encouraging results from experimental studies. A new approach is represented by the use of CAR-T cells engineered in vivo using lipid nanoparticles containing mRNA coding for a receptor directed against the FAP protein, expressed by cardiac myofibroblasts. This strategy has proved to be safe and effective in reducing myocardial fibrosis and improving cardiac function in mouse models of cardiac fibrosis. Clinical studies are required to test this novel approach in humans.
Cardiac tissue engineering, which combines cells and supportive scaffolds, is an emerging treatment for restoring cardiac function after myocardial infarction (MI), although, the optimal construct ...remains a challenge. We developed two engineered cardiac grafts, based on decellularized scaffolds from myocardial and pericardial tissues and repopulated them with adipose tissue mesenchymal stem cells (ATMSCs). The structure, macromechanical and micromechanical scaffold properties were preserved upon the decellularization and recellularization processes, except for recellularized myocardium micromechanics that was ∼2-fold stiffer than native tissue and decellularized scaffolds. Proteome characterization of the two acellular matrices showed enrichment of matrisome proteins and major cardiac extracellular matrix components, considerably higher for the recellularized pericardium. Moreover, the pericardial scaffold demonstrated better cell penetrance and retention, as well as a bigger pore size. Both engineered cardiac grafts were further evaluated in pre-clinical MI swine models. Forty days after graft implantation, swine treated with the engineered cardiac grafts showed significant ventricular function recovery. Irrespective of the scaffold origin or cell recolonization, all scaffolds integrated with the underlying myocardium and showed signs of neovascularization and nerve sprouting. Collectively, engineered cardiac grafts -with pericardial or myocardial scaffolds- were effective in restoring cardiac function post-MI, and pericardial scaffolds showed better structural integrity and recolonization capability.
Compelling evidence supports the therapeutic benefit of extracellular vesicles (EVs). EVs are nanostructures with a lipid bilayer membrane that are secreted by multiple cells, including mesenchymal ...stromal cells (MSCs), as means of cellular communication. MSC-EVs, resembling their MSC origin, carry protected immunomodulatory and pro-regenerative cargoes to targeted neighboring or distant cells and tissues. Though treatments focused on MSC-EVs have emerged as greatly versatile approaches to modulate multiple inflammatory-related conditions, crucial concerns, including the possibility of increasing therapeutic outcomes by pre-conditioning parental MSCs or engineering derived EVs and clarification of the most relevant mechanisms of action, remain. Here, we summarize the large amount of preclinical research surrounding the modulation of beneficial effects by MSC-EVs.
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•MSC-EV are a potential therapy through immunomodulation in multiple disease models.•Common MSC-EV mechanisms of action affect the NFκB and Stat3 pathways.•A variety of miRNA and EV-associated proteins account for foremost MSC-EV effects.•The bioengineering of MSC-EVs is a feasible option to enhance MSC-EV function.
Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord ...mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect.
Primary ventricular fibrillation (PVF) is a life-threatening complication of ST-segment elevation myocardial infarction (STEMI). It is unclear what roles viral infection and/or systemic inflammation ...may play as underlying triggers of PVF, as a second hit in the context of acute ischaemia. Here we aimed to evaluate whether the circulating virome and inflammatory proteome were associated with PVF development in patients with STEMI. Blood samples were obtained from non-PVF and PVF STEMI patients at the time of primary PCI, and from non-STEMI healthy controls. The virome profile was analysed using VirCapSeq-VERT (Virome Capture Sequencing Platform for Vertebrate Viruses), a sequencing platform targeting viral taxa of 342,438 representative sequences, spanning all virus sequence records. The inflammatory proteome was explored with the Olink inflammation panel, using the Proximity Extension Assay technology. After analysing all viral taxa known to infect vertebrates, including humans, we found that non-PVF and PVF patients only significantly differed in the frequencies of viruses in the Gamma-herpesvirinae and Anelloviridae families. In particular, most showed a significantly higher relative frequency in non-PVF STEMI controls. Analysis of systemic inflammation revealed no significant differences between the inflammatory profiles of non-PVF and PVF STEMI patients. Inflammatory proteins associated with cell adhesion, chemotaxis, cellular response to cytokine stimulus, and cell activation proteins involved in immune response (IL6, IL8 CXCL-11, CCL-11, MCP3, MCP4, and ENRAGE) were significantly higher in STEMI patients than non-STEMI controls. CDCP1 and IL18-R1 were significantly higher in PVF patients compared to healthy subjects, but not compared to non-PVF patients. The circulating virome and systemic inflammation were not associated with increased risk of PVF development in acute STEMI. Accordingly, novel strategies are needed to elucidate putative triggers of PVF in the setting of acute ischaemia, in order to reduce STEMI-driven sudden death burden.
Rationale: Extracellular vesicles (EVs) from mesenchymal stromal cell (MSC) are a potential therapy for cardiac healing after myocardial infarction (MI). Nevertheless, neither their efficient ...administration nor therapeutic mechanisms are fully elucidated. Here, we evaluate the preclinical efficacy of a tissue engineering approach to locally deliver porcine cardiac adipose tissue MSC-EV (cATMSC-EV) in an acute MI pig model.Methods: After MI by permanent ligation of the coronary artery, pigs (n = 24) were randomized to Untreated or treated groups with a decellularised pericardial scaffold filled with peptide hydrogel and cATMSC-EV purified by size exclusion chromatography (EV-Treated group) or buffer (Control group), placed over the post-infarcted myocardium.Results: After 30 days, cardiac MRI showed an improved cardiac function in EV-Treated animals, with significantly higher right ventricle ejection fraction (+20.8% in EV-Treated; p = 0.026), and less ventricle dilatation, indicating less myocardial remodelling. Scar size was reduced, with less fibrosis in the distal myocardium (-42.6% Col I in EV-Treated vs Untreated; p = 0.03), a 2-fold increase in vascular density (EV-Treated; p = 0.019) and less CCL2 transcription in the infarct core. EV-treated animals had less macrophage infiltration in the infarct core (-31.7% of CD163+ cells/field in EV-Treated; p = 0.026), but 5.8 times more expressing anti-inflammatory CD73 (p = 0.015). Systemically, locally delivered cATMSC-EV also triggered a systemic effect, doubling the circulating IL-1ra (p = 0.01), and reducing the PBMC rush 2d post-MI, the TNFα and GM-CSF levels at 30d post-MI, and modulating the CD73+ and CCR2+ monocyte populations, related to immunomodulation and fibrosis modulation.Conclusions: These results highlight the potential of cATMSC-EV in modulating hallmarks of ischemic injury for cardiac repair after MI.
Electrocardiogram (ECG) synchronization is useful to avoid the effects of cardiac motion in medical measurements, and is widely used in standard medical imaging. A number of medical equipment include ...embedded commercial synchronizers. However, the use of independent synchronization modules is sometimes needed when several non-integrated instruments are used, or in the development of new medical instruments and procedures. We present a simple low-cost ECG synchronizer module based on an Arduino controller board that converts the ECG signal into a transistor-transistor-logic (TTL) one, allowing real-time medical measurements triggered at specific phases of the cardiac cycle. The device and conversion algorithm developed is optimized in vitro using synthetic and human ECG signals, and tested in vivo on three swine specimens. Error rates during the in vivo testing stage remain below the 2% of the cycles in all animals and critical false positives are less than 1%, which is sufficient for most applications. Possible algorithm updates are discussed if its performance needs to be improved.
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