Development, characterization and evaluation of advanced therapies for the treatment of cardiac pathologies

Cardiovascular diseases (CVDs) are the leading cause of disease burden and mortality in the world, as well as a major cause of disability and health care costs. With the average lifespan of the human population continuously increasing, it is expected that the problem of CVDs will only continue to grow in the following years. Current pharmacological treatments for age-associated cardiac pathologies such as heart failure and atrial fibrillation present severe clinical efficacy and safety problems and are not regarded as definitive cures. This makes it necessary to develop new treatment strategies that target the involved molecular pathways and trigger endogenous reparative responses. Contrary to current molecular treatments, advanced therapy medicinal products (ATMPs) such as stem cells, extracellular vesicles (EVs) and biomaterials such as hydrogels could have the potential to treat cardiac aging-associated pathologies from a more fundamental level. However, many problems and unknowns still need to be solved before they can reach the clinical scenario. Some of the most highlighted limitations we focus on in this work are: (i) the lack of deep understanding of their mechanism of action (MoA), (ii) their large variability and lack of standardization (including inadequate potency tests) and (iii) low in vivo retention at the site of interest. Therefore, the main objective of this thesis is to develop, characterize and evaluate advanced therapies for the treatment of cardiac pathologies solving some of their current limitations to enhance their therapeutic potential. To achieve this aim, we first focus on improving standardization and development of potency assays. We describe the main characteristics and challenges for a cell therapy based potency test in the cardiovascular field and we review and propose different types of assays that could be taken into consideration based on the product’s expected MoA and the target cardiovascular disease. Secondly, as cardiosphere-derived cells (CDCs) and their secreted EVs (CDC-EVs) have previously reported to have anti-senescent effects and this is considered important in aging-related cardiac diseases, we explore potential predictors of rejuvenating potency with a special focus on the chronological age of the CDC-donors and CDC-senescence, among others. Multiple in vitro tests allow us to conclude that more than cell particular biological markers or characteristics, the cell bioactivity relative to the expected MoA should be a better predictor for the ATMP potency. Thus, we evaluate if the in vitro anti-senescent and pro-angiogenic effect of the CDC-EVs, scored with a matrix assay, can be used to predict the in vivo potency of the CDC-EVs in an animal model of cardiac aging. Our results show that EVs classified in vitro as potent with the matrix assay have more cardiac reparative potential in vivo than EVs classified as non-potent. After further validation, the matrix assay proposed here could be a suitable in vitro potency test for discerning suitable allogenic biological products in the cardiac aging clinical scenario. Next, with the purpose of improving EV retention at the site of interest, we develop an optimized product combining hydrogels from cardiac extracellular matrix (cECM), polyethylene glycol and EVs to overcome some of their individual limitations: long gelation time of the cECM and poor retention of the EVs. We conclude that the combined product rapidly gels at physiological temperature and presents improved mechanical properties while maintaining the injectability, the biodegradability, and the bioactivity of its individual components. In addition, it serves to better retain the EVs on-site in vivo. Finally, we explore the electrophysiological modifications induced by CDC-EVs on arrhythmogenic tissue to better understand the mechanisms behind their antiarrhythmic effect. We found that CDC-EVs reduce spontaneous activation complexity and increase conduction velocity of cardiomyocytes leading to a less arrhythmogenic profile. If validated in other cellular models, CDC-EVs may be used specifically as antiarrhythmic agents in a wide range of cardiac pathologies. Although future work should aim to further validate these results both at preclinical and clinical level, these findings together partially overcome some of the main challenges for the therapeutic use of cellular therapies and open a new horizon for the treatment of cardiac-aging related pathologies, some still considered as unmet medical needs.Programa de Doctorado en Ciencia y Tecnología Biomédica por la Universidad Carlos III de MadridPresidenta: Eva Delpón Mosquera.- Secretaria: Marta García Díez.- Vocal: Javier Bermejo Thoma

Characterization of the effect of the substrate over functional and electrophysiological properties during culture of cardiomyocytes

Heart failure is a common, costly and potentially fatal condition commonly caused by cardiac cells depletion. Current therapies are aimed at protecting surviving cardiomyocytes, but they are unable to produce cardiac regeneration. Tissue engineered cardiac patches seem promising as cardiac repair tools, but they still pose many limitations as they do not mimic in vivo behavior. Recently, the use of flexible membranes for cardiac cell culture has been suggested as determinant in cardiomyocytic properties. The goal of this study is to compare for the first time key cardiomyocyte functional properties for cardiac repair such as proliferation, migration and displacement, and electrophysiological properties of HL-1 cardiac cell line in two different materials: (1) rigid Petri dishes and (2) flexible PDMS (polydimethylsiloxane) silicon wells. The study was carried out in the Laboratorio de Órganos y Matrices Bioartificiales belonging to the Instituto de Investigación Sanitaria Gregorio Marañón. In order to assess these properties, HL-1 cells were cultured on both substrates and different tests were performed. Proliferation assay was carried out using alamarBlue® colorimetric test to calculate the percentage of reduction which is directly related to cell proliferation. Migration and displacement were determined by performing wound tests followed up using time-lapse imaging, and quantified using custom software made in Matlab. To study characteristic properties of cardiomyocytes, impulse propagation was recorded using optical mapping techniques, and results were analysed using custom software developed in Matlab to obtain conduction velocity information. At molecular level, expression of genes coding for proteins involved in impulse generation and propagation was analysed using qPCR (quantitative Polymerase Chain Reaction) technique. Results show that HL-1 cells were able to grow and retain an adult cardiomyocyte phenotype on both substrates and spontaneous contractile activity was kept. In general, proliferation of HL-1 cells was confirmed to be very high, in opposite to migration and displacement. Proliferation was higher at early stages in Petri, but as the culture grew, proliferation rate got higher in silicon. Migration and displacement were quite low as wounds did not close within 60 hours, which is characteristic of adult cardiomyocytes. The results and observations suggest that these cells colonize new areas only by proliferation. Regarding the electrophysiological properties, conduction velocity showed to be higher in silicon wells (150 %). This result was supported by a higher expression of genes involved in action potential. Results from this study support our hypothesis that the use of flexible membranes induces a more similar cardiac phenotype to that shown in vivo, especially characterized by higher conduction velocities. This opens new insights into cardiac patches, confirming that mechanical characteristics of the substrate play a role in cardiac cell phenotype. Silicon wells and other flexible membranes will allow further mimicking the in vivo environment by growing cardiac cells under mechanical and electrical stimulation.Ingeniería Biomédic

Electrophysiological effects of extracellular vesicles secreted by cardiosphere-derived cells: Unraveling the antiarrhythmic properties of cell therapies

This article belongs to the Special Issue Advances in Regenerative Medicine and Tissue Engineering.Although cell-based therapies show potential antiarrhythmic effects that could be mediated by their paracrine action, the mechanisms and the extent of these effects were not deeply explored. We investigated the antiarrhythmic mechanisms of extracellular vesicles secreted by cardiosphere-derived cell extracellular vesicles (CDC-EVs) on the electrophysiological properties and gene expression profile of HL1 cardiomyocytes. HL-1 cultures were primed with CDC-EVs or serum-free medium alone for 48 h, followed by optical mapping and gene expression analysis. In optical mapping recordings, CDC-EVs reduced the activation complexity of the cardiomyocytes by 40%, increased rotor meandering, and reduced rotor curvature, as well as induced an 80% increase in conduction velocity. HL-1 cells primed with CDC-EVs presented higher expression of SCN5A, CACNA1C, and GJA1, coding for proteins involved in INa, ICaL, and Cx43, respectively. Our results suggest that CDC-EVs reduce activation complexity by increasing conduction velocity and modifying rotor dynamics, which could be driven by an increase in expression of SCN5A and CACNA1C genes, respectively. Our results provide new insights into the antiarrhythmic mechanisms of cell therapies, which should be further validated using other models.This research was funded by the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain: PI16/01123, PI17/01059, Red de Terapia Celular-Tercel-RD16.0011.0029 and CIBERCV-CB16.11.00292

Cardiac Extracellular Matrix Hydrogel Enriched with Polyethylene Glycol Presents Improved Gelation Time and Increased On-Target Site Retention of Extracellular Vesicles

Stem-cell-derived extracellular vesicles (EVs) have demonstrated multiple beneficial effects in preclinical models of cardiac diseases. However, poor retention at the target site may limit their therapeutic efficacy. Cardiac extracellular matrix hydrogels (cECMH) seem promising as drug-delivery materials and could improve the retention of EVs, but may be limited by their long gelation time and soft mechanical properties. Our objective was to develop and characterize an optimized product combining cECMH, polyethylene glycol (PEG), and EVs (EVs–PEG–cECMH) in an attempt to overcome their individual limitations: long gelation time of the cECMH and poor retention of the EVs. The new combined product presented improved physicochemical properties (60% reduction in half gelation time, p < 0.001, and threefold increase in storage modulus, p < 0.01, vs. cECMH alone), while preserving injectability and biodegradability. It also maintained in vitro bioactivity of its individual components (55% reduction in cellular senescence vs. serum-free medium, p < 0.001, similar to EVs and cECMH alone) and increased on-site retention in vivo (fourfold increase vs. EVs alone, p < 0.05). In conclusion, the combination of EVs–PEG–cECMH is a potential multipronged product with improved gelation time and mechanical properties, increased on-site retention, and maintained bioactivity that, all together, may translate into boosted therapeutic efficacy

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Electrophysiological Effects of Extracellular Vesicles Secreted by Cardiosphere-Derived Cells: Unraveling the Antiarrhythmic Properties of Cell Therapies

Although cell-based therapies show potential antiarrhythmic effects that could be mediated by their paracrine action, the mechanisms and the extent of these effects were not deeply explored. We investigated the antiarrhythmic mechanisms of extracellular vesicles secreted by cardiosphere-derived cell extracellular vesicles (CDC-EVs) on the electrophysiological properties and gene expression profile of HL1 cardiomyocytes. HL-1 cultures were primed with CDC-EVs or serum-free medium alone for 48 h, followed by optical mapping and gene expression analysis. In optical mapping recordings, CDC-EVs reduced the activation complexity of the cardiomyocytes by 40%, increased rotor meandering, and reduced rotor curvature, as well as induced an 80% increase in conduction velocity. HL-1 cells primed with CDC-EVs presented higher expression of SCN5A, CACNA1C, and GJA1, coding for proteins involved in INa, ICaL, and Cx43, respectively. Our results suggest that CDC-EVs reduce activation complexity by increasing conduction velocity and modifying rotor dynamics, which could be driven by an increase in expression of SCN5A and CACNA1C genes, respectively. Our results provide new insights into the antiarrhythmic mechanisms of cell therapies, which should be further validated using other models

The Essential Need for a Validated Potency Assay for Cell-Based Therapies in Cardiac Regenerative and Reparative Medicine. A Practical Approach to Test Development

Biological treatments are one of the medical breakthroughs in the twenty-first century. The initial enthusiasm pushed the field towards indiscriminatory use of cell therapy regardless of the pathophysiological particularities of underlying conditions. In the reparative and regenerative cardiovascular field, the results of the over two decades of research in cell-based therapies, although promising still could not be translated into clinical scenario. Now, when we identified possible deficiencies and try to rebuild its foundations rigorously on scientific evidence, development of potency assays for the potential therapeutic product is one of the steps which will bring our goal of clinical translation closer. Although, highly challenging, the potency tests for cell products are considered as a priority by the regulatory agencies. In this paper we describe the main characteristics and challenges for a cell therapy potency test focusing on the cardiovascular field. Moreover, we discuss different steps and types of assays that should be taken into consideration for an eventual potency test development by tying together two fundamental concepts: target disease and expected mechanism of action

Cardiovascular diseases in the digital health era: A translational approach from the lab to the clinic

This article belongs to the Special Issue Bioinformatics: Present and Future BiotechnologyTranslational science has been introduced as the nexus among the scientific and the clinical field, which allows researchers to provide and demonstrate that the evidence-based research can connect the gaps present between basic and clinical levels. This type of research has played a major role in the field of cardiovascular diseases, where the main objective has been to identify and transfer potential treatments identified at preclinical stages into clinical practice. This transfer has been enhanced by the intromission of digital health solutions into both basic research and clinical scenarios. This review aimed to identify and summarize the most important translational advances in the last years in the cardiovascular field together with the potential challenges that still remain in basic research, clinical scenarios, and regulatory agencies.Supported in part by the Instituto de Salud Carlos III (PI16/01123, DTS16/0160, PI17/01059, PI19/00161, PI20/01618, DTS21/00064 and PI18/01895, Red TERCEL RETIC RD16/0011/0029) and the project RICORS "RD21/0017; TERAV" (Group: 002). The Regional Government of Madrid (S2017/BMD-3962, Avancell-CM); Spanish Ministry of Science and Innovation (CIBERCV), and European Union's H2020 Program under grant agreement No. 874827 (BRAV3)