Gene transfer-based strategies for heart regeneration

Ischemic heart disease is the leading cause of mortality worldwide. Its most frequent and serious complication is acute myocardial infarction, after which the remaining contractile tissue undergoes a process characterized by myocyte hypertrophy and death of the remaining myocardium, and its progressive replacement by fibrosis (Sutton and Sharpe 2000). This process, termed remodeling, eventually leads to left ventricular dilation and heart failure. The extent of remodeling is largely dependent upon infarct size (Lenderink et al. 1995). Small infarcts do not induce significant remodeling. Large ones, contrarily, provoke substantial remodeling and therefore evolve towards contractile failure. This explains why regenerating the contractile tissue, a process named cardiomyogenesis, has become a major objective in biomedical research.Sociedad Argentina de Fisiologí

Lights and shadows in cardiac regeneration

Given that the adult human heart has an extremely limited regenerative capacity, diseases characterized by contractile cell loss, as myocardial infarction and cardiomyopathies, lead to ventricular remodeling and heart failure. Hence, diverse strategies to promote myocardial regeneration have been proposed and assessed in animals and humans with ischemic heart disease. Of these, gene transfer and especially stem cell therapy have been used. So far, the overall main outcome is a gross disparity between the promising results obtained in mammalian models and the poor, if any, benefit observed in randomized, controlled clinical trials. Many reasons may account for this disappointing scenario. Some, including flawed trial design and methodology, differences in cell type and dosing as well as in route of administration, erroneous end points selection and heterogeneous patient populations have been extensively discussed in comprehensive reviews. Others, more recently addressed, signal the use of inadequate or non-precise laboratory techniques in cell identification and fate, this leading to precarious or misleading conclusions. We hereby summarize part of the work done and quote some new approaches, like the use of induced pluripotent stem cells and the promotion of selfregeneration by targeting the adult cardiomyocyte cell cycle, that may cast some light in the otherwise shadowy field of cardiac regeneration.Dada la limitadísima capacidad regenerativa del corazón humano adulto, las enfermedades caracterizadas por pérdida de tejido contráctil, como el infarto de miocardio y las miocardiopatías, conducen al remodelamiento ventricular y la insuficiencia cardíaca. Por ello, diversas estrategias cardiorregenerativas han sido propuestas y evaluadas en modelos animales y pacientes con cardiopatía isquémica. De ellas, las más usadas han sido la transferencia génica y, especialmente, la terapia con células madre. Hasta aquí, el resultado global es una gran disparidad entre los prometedores resultados obtenidos en modelos animales y los pobres o nulos beneficios observados en los ensayos clínicos. Muchas razones explican este decepcionante escenario. Algunas, tales como imperfecciones de diseño y metodología, diferencias en el tipo y dosis de células así como en la vía de administración, puntos finales erróneamente elegidos, y heterogeneidad en las poblaciones de pacientes, han sido ampliamente discutidos en muy completas revisiones. Otros, más recientemente abordados, señalan el uso de inadecuadas o imprecisas técnicas de laboratorio para identificar el tipo de célula y su destino, conducentes a conclusiones precarias o engañosas. En este artículo resumimos parte del trabajo realizado y citamos algunos nuevos abordajes, tales como el uso de células pluripotentes inducidas y la auto- regeneración por manipulación del ciclo celular del miocardiocito adulto, que podrían arrojar algo de luz al sombrío campo de la regeneración cardíaca.Sociedad Argentina de Fisiologí

In silico performance analysis of web tools for CRISPRa sgRNA design in human genes

Angiogenic gene overexpression has been the main strategy in numerous vascular regenerative gene therapy projects. However, most have failed in clinical trials. CRISPRa technology enhances gene overexpression levels based on the identification of sgRNAs with maximum efficiency and safety. CRISPick and CHOP CHOP are the most widely used web tools for the prediction of sgRNAs. The objective of our study was to analyze the performance of both platforms for the sgRNA design to angiogenic genes (VEGFA, KDR, EPO, HIF-1A, HGF, FGF, PGF, FGF1) involving different human reference genomes (GRCH 37 and GRCH 38). The top 20 ranked sgRNAs proposed by the two tools were analyzed in different aspects. No significant differences were found on the DNA curvature associated with the sgRNA binding sites but the sgRNA predicted on-target efficiency was significantly greater when CRISPick was used. Moreover, the mean ranking variation was greater for the same platform in EPO, EGF, HIF-1A, PGF and HGF, whereas it did not reach statistical significance in KDR, FGF-1 and VEGFA. The rearrangement analysis of the ranking positions was also different between platforms. CRISPick proved to be more accurate in establishing the best sgRNAs in relation to a more complete genome, whereas CHOP CHOP showed a narrower classification reordering.Fil: Nuñez Pedrozo, Cristian Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Peralta, Tomás M.. Universidad Favaloro; ArgentinaFil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Locatelli, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Crottogini, Alberto Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Belaich, Mariano Nicolas. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cuniberti, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentin

An ovine model of postinfarction dilated cardiomyopathy in animals with highly variable coronary anatomy

Studies on cardiac regeneration require large mammalian models of dilated cardiomyopathy (DCM) after acute myocardial infarction (AMI), and pig and sheep models are increasingly used in this field of preclinical research. Given the large interindividual variability in ovine left anterior descending artery (LAD) anatomy, protocols based on the coronary arteries to be ligated often lead to significant variation in infarct sizes and hence to heterogeneous results, ranging from no ventricular remodeling to acute, lethal left ventricular (LV) failure. We designed an ovine model of postinfarction DCM based on estimated infarct size rather than on a predetermined menu of coronary artery ligatures. In seven adult sheep we induced an anterolateral AMI of approximately 25% of the LV mass by ligating the branches of the LAD that, by visual inspection, would lead to such an infarct size. In 10 to 12 weeks, LV end-diastolic volume more than doubled and LV end-systolic volume almost tripled. LV ejection fraction decreased dramatically, as did LV percent fractional shortening and LV percent wall thickening. Infarct size (planimetry) was approximately 25% of the LV endocardial surface. We conclude that in sheep, an anterolateral AMI of approximately 25% of the LV mass--regardless of the coronary branches ligated to attain that infarct size--results in a model of postinfarction DCM that may prove useful in preclinical research on myocardial regeneration.Fil: Locatelli, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; ArgentinaFil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; ArgentinaFil: Mendiz, Oscar. Fundación Favaloro; ArgentinaFil: Salmo, Fabián. Fundación Favaloro; ArgentinaFil: Fazzi, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Favaloro; ArgentinaFil: Hnatiuk, Anna. Universidad Favaloro; ArgentinaFil: Laguens, Rubén. Fundación Favaloro; ArgentinaFil: Crottogini, Alberto Jose. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Favaloro; Argentin

Allogeneic mesenchymal stromal cells overexpressing mutant human Hypoxia-inducible factor 1-α (HIF1-α) in an ovine model of acute myocardial infarction

Background-Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia-inducible factor 1-α (HIF1-α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen-resistant HIF1-α would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI. Methods and Results-Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1-α (BMMSC-HIF) were injected in the peri-infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P < 0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2-fold (P < 0.001) in the presence of markedly decreased end-systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo-treated animals (n=6), neither parameters changed over time. HIF1-α-transfected BMMSCs (BMMSC-HIF) induced angio-/arteriogenesis and decreased apoptosis by HIF1-mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin-1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long-term retention of BMMSC-HIF. Conclusions-Intramyocardial delivery of BMMSC-HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1-mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.Fil: Hnatiuk, Anna. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Ong, Sang-Ging. Stanford University School of Medicine; Estados UnidosFil: Olea, Fernanda Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Locatelli, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Riegler, Johannes. Stanford University School of Medicine; Estados UnidosFil: Lee, Won Hee. Stanford University School of Medicine; Estados UnidosFil: Jen, Cheng Hao. University of London; Reino UnidoFil: De Lorenzi, Andrea. Fundación Favaloro; ArgentinaFil: Giménez, Carlos Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Laguens, Rubén. Universidad Favaloro; ArgentinaFil: Wu, Joseph C.. Stanford University School of Medicine; Estados UnidosFil: Crottogini, Alberto José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentin

Effect of vascular endothelial growth factor gene transfer on infarct size, left ventricular function and myocardial perfusion in sheep after 2months of coronary artery occlusion

Background: In large mammalian models of acute myocardial infarction (AMI), plasmid-mediated vascular endothelial growth factor (pVEGF) gene transfer has been shown to induce angio-arteriogenesis, proliferation of myocyte precursors and adult cardiomyocyte mitosis, reducing infarct size at 15days after coronary artery occlusion. However, it is unknown whether these effects persist at longer follow-up times, nor how they affect cardiac performance. We thus assessed infarct size, left ventricular (LV) function and perfusion in 2-month-old ovine AMI. Methods: Adult sheep with coronary artery occlusion were randomized to blindly receive ten intramyocardial injections of 3.8mg of pVEGF or empty plasmid distributed at the infarct border. Three and 60days later, LV perfusion (single-photon emission computed tomography) and function (stress echocardiography) were assessed. Finally, hemodynamics (LV catheterization), scar size and peri-infarct histology were studied. Results: Infarct size was 30% smaller in pVEGF-treated sheep (23.6±1.9% versus 32.7±2.7% of the LV; p<0.02). Percentage fractional shortening and wall thickening at the infarct border improved after pVEGF, as did myocardial perfusion and LV wall motion under pharmacological stress. Global LV function did not differ between groups, although the force-frequency response was preserved in pVEGF group and lost in placebo animals. These effects were associated with angio-arteriogenesis and proliferation of cardiomyocyte precursors. Conclusions: In sheep with AMI, pVEGF gene transfer affords long-term infarct size reduction, yielding regional LV function and perfusion improvement and reducing remodeling progression. These results suggest the potential usefulness of this approach in the clinical setting.Fil: Vera Janavel, Gustavo L.. Universidad Favaloro. Área de Investigación y Desarrollo; ArgentinaFil: De Lorenzi, Andrea. Fundación Favaloro; ArgentinaFil: Cortés, Claudia. Fundación Favaloro; ArgentinaFil: Olea, Fernanda Daniela. Universidad Favaloro. Área de Investigación y Desarrollo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cabeza Meckert, Patricia. Fundación Favaloro; ArgentinaFil: Bercovich, Andrés. Biosidus S. A.; ArgentinaFil: Criscuolo, Marcelo. Biosidus S. A.; ArgentinaFil: Laguens, Rubén. Universidad Favaloro. Área de Investigación y Desarrollo; ArgentinaFil: Crottogini, Alberto Jose. Universidad Favaloro. Área de Investigación y Desarrollo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Activated macrophages as a feeder layer for growth of resident cardiac progenitor cells

The adult heart contains a population of cardiac progenitor cells (CPCs). Growing and collecting an adequate number of CPCs demands complex culture media containing growth factors. Since activated macrophages secrete many growth factors, we investigated if activated isolated heart cells seeded on a feeder layer of activated peritoneal macrophages (PM) could result in CPCs and if these, in turn, could exert cardioprotection in rats with myocardial infarction (MI). Heart cells of inbred Wistar rats were isolated by collagenase digestion and cultured on PM obtained 72 h after intraperitoneal injection of 12 ml thioglycollate. Cells (1 × 106) exhibiting CPC phenotype (immunohistochemistry) were injected in the periphery of rat MI 10 min after coronary artery occlusion. Control rats received vehicle. Three weeks later, left ventricular (LV) function (echocardiogram) was assessed, animals were euthanized and the hearts removed for histological studies. Five to six days after seeding heart cells on PM, spherical clusters composed of small bright and spherical cells expressing mostly c-Kit and Sca-1 antigens were apparent. After explant, those clusters developed cobblestone-like monolayers that expressed smooth muscle actin and sarcomeric actin and were successfully transferred for more than ten passages. When injected in the MI periphery, many of them survived at 21 days after coronary ligature, improved LV ejection fraction and decreased scar size as compared with control rats. CPC-derived cells with cardiocyte and smooth muscle phenotypes can be successfully grown on a feeder layer of activated syngeneic PM. These cells decreased scar size and improved heart function in rats with MI.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Aligned ovine diaphragmatic myoblasts overexpressing human connexin-43 seeded on poly (L-lactic acid) scaffolds for potential use in cardiac regeneration

Diaphragmatic myoblasts (DMs) are precursors of type-1 muscle cells displaying high exhaustion threshold on account that they contract and relax 20 times/min over a lifespan, making them potentially useful in cardiac regeneration strategies. Besides, it has been shown that biomaterials for stem cell delivery improve cell retention and viability in the target organ. In the present study, we aimed at developing a novel approach based on the use of poly (L-lactic acid) (PLLA) scaffolds seeded with DMs overexpressing connexin-43 (cx43), a gap junction protein that promotes inter-cell connectivity. DMs isolated from ovine diaphragm biopsies were characterized by immunohistochemistry and ability to differentiate into myotubes (MTs) and transduced with a lentiviral vector encoding cx43. After confirming cx43 expression (RT-qPCR and Western blot) and its effect on inter-cell connectivity (fluorescence recovery after photobleaching), DMs were grown on fiberaligned or random PLLA scaffolds. DMs were successfully isolated and characterized. Cx43 mRNA and protein were overexpressed and favored inter-cell connectivity. Alignment of the scaffold fibers not only aligned but also elongated the cells, increasing the contact surface between them. This novel approach is feasible and combines the advantages of bioresorbable scaffolds as delivery method and a cell type that on account of its features may be suitable for cardiac regeneration. Future studies on animal models of myocardial infarction are needed to establish its usefulness on scar reduction and cardiac function.Centro de Investigaciones Cardiovasculare

Decreasing hospital burden of COVID-19 during the first wave in Regione Lombardia: an emergency measures context

Abstract: Background: The aim of this study is to quantify the hospital burden of COVID-19 during the first wave and how it changed over calendar time; to interpret the results in light of the emergency measures introduced to manage the strain on secondary healthcare. Methods: This is a cohort study of hospitalised confirmed cases of COVID-19 admitted from February–June 2020 and followed up till 17th July 2020, analysed using a mixture multi-state model. All hospital patients with confirmed COVID-19 disease in Regione Lombardia were involved, admitted from February–June 2020, with non-missing hospital of admission and non-missing admission date. Results: The cohort consists of 40,550 patients hospitalised during the first wave. These patients had a median age of 69 (interquartile range 56–80) and were more likely to be men (60%) than women (40%). The hospital-fatality risk, averaged over all pathways through hospital, was 27.5% (95% CI 27.1–28.0%); and steadily decreased from 34.6% (32.5–36.6%) in February to 7.6% (6.3–10.6%) in June. Among surviving patients, median length of stay in hospital was 11.8 (11.6–12.3) days, compared to 8.1 (7.8–8.5) days in non-survivors. Averaged over final outcomes, median length of stay in hospital decreased from 21.4 (20.5–22.8) days in February to 5.2 (4.7–5.8) days in June. Conclusions: The hospital burden, in terms of both risks of poor outcomes and lengths of stay in hospital, has been demonstrated to have decreased over the months of the first wave, perhaps reflecting improved treatment and management of COVID-19 cases, as well as reduced burden as the first wave waned. The quantified burden allows for planning of hospital beds needed for current and future waves of SARS-CoV-2 i

Decreasing hospital burden of COVID-19 during the first wave in Regione Lombardia: an emergency measures context

Abstract: Background: The aim of this study is to quantify the hospital burden of COVID-19 during the first wave and how it changed over calendar time; to interpret the results in light of the emergency measures introduced to manage the strain on secondary healthcare. Methods: This is a cohort study of hospitalised confirmed cases of COVID-19 admitted from February–June 2020 and followed up till 17th July 2020, analysed using a mixture multi-state model. All hospital patients with confirmed COVID-19 disease in Regione Lombardia were involved, admitted from February–June 2020, with non-missing hospital of admission and non-missing admission date. Results: The cohort consists of 40,550 patients hospitalised during the first wave. These patients had a median age of 69 (interquartile range 56–80) and were more likely to be men (60%) than women (40%). The hospital-fatality risk, averaged over all pathways through hospital, was 27.5% (95% CI 27.1–28.0%); and steadily decreased from 34.6% (32.5–36.6%) in February to 7.6% (6.3–10.6%) in June. Among surviving patients, median length of stay in hospital was 11.8 (11.6–12.3) days, compared to 8.1 (7.8–8.5) days in non-survivors. Averaged over final outcomes, median length of stay in hospital decreased from 21.4 (20.5–22.8) days in February to 5.2 (4.7–5.8) days in June. Conclusions: The hospital burden, in terms of both risks of poor outcomes and lengths of stay in hospital, has been demonstrated to have decreased over the months of the first wave, perhaps reflecting improved treatment and management of COVID-19 cases, as well as reduced burden as the first wave waned. The quantified burden allows for planning of hospital beds needed for current and future waves of SARS-CoV-2 i