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Myocardial infarction is a prevalent cardiovascular disease. Mechanisms of repair in the post-infarcted heart include a progressive fibrosis that severely affects cardiac performance, eventually leading to cardiac failure. Cardiac fibrosis in the context of ventricular remodeling after infarction depends on fibroblasts of the cardiac interstitium (cardiac fibroblasts), a heterogeneous population of cells which, upon interaction with other interstitial cell types, initiates a massive deposition of extracellular matrix promoting the formation of a characteristic scar. In this work we have studied the cellular components of the cardiac interstitium from the embryo to the adult. Our results show that Wilms tumor supresor (Wt1) positive epicardial-derived mesenchymal cells pioneer the formation of the cardiac interstitium along embryogenesis, followed by the peri- and post-natal incorporation of bone-marrow derived cells. Adult epicardial-derived cells robustly differentiate into cardiac fibroblasts under normal and pathologic conditions, and become the predominant fibroblast type in the post-infarction scar. Furthermore, epicardial-derived cardiac fibroblasts are shown to display stromal properties respect to bone marrow-derived cells, critically contributing to the homing and persistence of circulating cells after infarction. [ABSTRACT FROM PUBLISHER]

Cardiac fibrosis, a process characterized by excessive extracellular matrix (ECM) deposition, is a common pathological consequence of many cardiovascular diseases (CVDs) normally resulting in organ failure and death. Cardiac fibroblasts (CFs) play an essential role in deleterious cardiac remodeling and dysfunction. In response to injury, quiescent CFs become activated and adopt a collagen-secreting phenotype highly contributing to cardiac fibrosis. In recent years, studies have been focused on the exploration of molecular and cellular mechanisms implicated in the activation process of CFs, which allow the development of novel therapeutic approaches for the treatment of cardiac fibrosis. Transcriptomic analyses using single-cell RNA sequencing (RNA-seq) have helped to elucidate the high cellular diversity and complex intercellular communication networks that CFs establish in the mammalian heart. Furthermore, a significant body of work supports the critical role of epigenetic regulation on the expression of genes involved in the pathogenesis of cardiac fibrosis. The study of epigenetic mechanisms, including DNA methylation, histone modification, and chromatin remodeling, has provided more insights into CF activation and fibrotic processes. Targeting epigenetic regulators, especially DNA methyltransferases (DNMT), histone acetylases (HAT), or histone deacetylases (HDAC), has emerged as a promising approach for the development of novel anti-fibrotic therapies. This review focuses on recent transcriptomic advances regarding CF diversity and molecular and epigenetic mechanisms that modulate the activation process of CFs and their possible clinical applications for the treatment of cardiac fibrosis.

Generating organs from stem cells through blastocyst complementation is a promising approach to meet the clinical need for transplants. In order to generate rejection-free organs, complementation of both parenchymal and vascular cells must be achieved, as endothelial cells play a key role in graft rejection. Here, we used a lineage-specific cell ablation system to produce mouse embryos unable to form both the cardiac and vascular systems. By mouse intraspecies blastocyst complementation, we rescued heart and vascular system development separately and in combination, obtaining complemented hearts with cardiomyocytes and endothelial cells of exogenous origin. Complemented chimeras were viable and reached adult stage, showing normal cardiac function and no signs of histopathological defects in the heart. Furthermore, we implemented the cell ablation system for rat-to-mouse blastocyst complementation, obtaining xenogeneic hearts whose cardiomyocytes were completely of rat origin. These results represent an advance in the experimentation towards the in vivo generation of transplantable organs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the development of therapeutic extracellular vesicles (EVs), drug encapsulation efficiencies are significantly lower when compared with synthetic nanomedicines. This is due to the hierarchical structure of the EV membrane and the physicochemical properties of the candidate drug (molecular weight, hydrophilicity, lipophilicity, and so on). As a proof of concept, here we demonstrated the importance of drug compartmentalization in EVs as an additional parameter affecting the therapeutic potential of drug-loaded EVs. In human adipose mesenchymal stem cell (hADSC) derived EVs, we performed a comparative drug loading analysis using two formulations of the same chemotherapeutic molecule - free doxorubicin (DOX) and 1,2-distearoyl- sn-glycero -3-phosphoethanolamine (DSPE) lipid-conjugated doxorubicin (L-DOX) - to enhance the intracellular uptake and therapeutic efficacy. By nano surface energy transfer (NSET) and molecular simulation techniques, along with cryo-TEM analysis, we confirmed the differential compartmentalization of these two molecules in hADSC EVs. L-DOX was preferentially adsorbed onto the surface of the EV, due to its higher lipophilicity, whereas free DOX was mostly encapsulated within the EV core. Also, the L-DOX loaded EV (LDOX@EV) returned an almost three-fold higher DOX content as compared to the free DOX loaded EV (DOX@EV), for a given input mass of drug. Based on the cellular investigations, L-DOX@EV showed higher cell internalization than DOX@EV. Also, in comparison with free L-DOX, the magnitude of therapeutic potential enhancement displayed by the surface compartmentalized L-DOX@EV is highly promising and can be exploited to overcome the sensitivity of many potential drugs, which are impermeable in nature. Overall, this study illustrates the significance of drug compartmentalization in EVs and how this could affect intracellular delivery, loading efficiency, and therapeutic effect. This will further lay the foundation for the future systematic investigation of EV-based biotherapeutic delivery platforms for personalized medicine., Competing Interests: All authors declared no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Oxidative stress-induced myocardial apoptosis and necrosis are critically involved in ischemic infarction, and several sources of extracellular vesicles appear to be enriched in therapeutic activities. The central objective was to identify and validate the differential exosome miRNA repertoire in human cardiac progenitor cells (CPC). CPC exosomes were first analyzed by LC-MS/MS and compared by RNAseq with exomes of human mesenchymal stromal cells and human fibroblasts to define their differential exosome miRNA repertoire (exo-miR SEL ). Proteomics demonstrated a highly significant representation of cardiovascular development functions and angiogenesis in CPC exosomes, and RNAseq analysis yielded about 350 different miRNAs; among the exo-miR SEL population, miR-935 was confirmed as the miRNA most significantly up-regulated; interestingly, miR-935 was also found to be preferentially expressed in mouse primary cardiac Bmi1 +high CPC, a population highly enriched in progenitors. Furthermore, it was found that transfection of an miR-935 antagomiR combined with oxidative stress treatment provoked a significant increment both in apoptotic and necrotic populations, whereas transfection of a miR-935 mimic did not modify the response. Conclusion. miR-935 is a highly differentially expressed miRNA in exo-miR SEL , and its expression reduction promotes oxidative stress-associated apoptosis. MiR-935, together with other exosomal miRNA members, could counteract oxidative stress-related apoptosis, at least in CPC surroundings.

The deposition of stem cells at sites of injury is a clinically relevant approach to facilitate tissue repair and angiogenesis. However, insufficient cell engraftment and survival require the engineering of novel scaffolds. Here, a regular network of microscopic poly(lactic-co-glycolic acid) (PLGA) filaments was investigated as a promising biodegradable scaffold for human Adipose-Derived Stem Cell (hADSC) tissue integration. Via soft lithography, three different microstructured fabrics were realized where 5 × 5 and 5 × 3 μm PLGA 'warp' and 'weft' filaments crossed perpendicularly with pitch distances of 5, 10 and 20 μm. After hADSC seeding, cell viability, actin cytoskeleton, spatial organization and the secretome were characterized and compared to conventional substrates, including collagen layers. On the PLGA fabric, hADSC re-assembled to form spheroidal-like structures, preserving cell viability and favoring a nonlinear actin organization. Moreover, the secretion of specific factors involved in angiogenesis, the remodeling of the extracellular matrix and stem cell homing was favored on the PLGA fabric as compared to that which occurred on conventional substrates. The paracrine activity of hADSC was microstructure-dependent, with 5 μm PLGA fabric enhancing the expression of factors involved in all three processes. Although more studies are needed, the proposed PLGA fabric would represent a promising alternative to conventional collagen substrates for stem cell implantation and angiogenesis induction.

Several Cre recombinase transgenic mouse models have been generated for cardiac fibroblast (CF) tracking and heart regulation. However, there is still no consensus on the ideal mouse model to optimally identify and/or regulate these cells. Here, a comparative evaluation of the efficiency and specificity of the indirect reporter Cre-loxP system was carried out in three of the most commonly used fibroblast reporter transgenic mice (Pdgfr a -CreERT2, Col1a1-CreERT2 and PostnMCM) under healthy and ischemic conditions, to determine their suitability in in vivo studies of cardiac fibrosis. We demonstrate optimal Cre recombinase activity in CF (but also, although moderate, in endothelial cells (ECs)) derived from healthy and infarcted hearts in the PDGFR a -creERT2 mouse strain. In contrast, no positive reporter signal was found in CF derived from the Col1a1-CreERT2 mice. Finally, in the PostnMCM line, fluorescent reporter expression was specifically detected in activated CF but not in EC, which leads us to conclude that it may be the most reliable model for future studies on cardiovascular disease. Importantly, no lethality or cardiac fibrosis were induced after tamoxifen administration at the established doses, either in healthy or infarcted mice of the three fibroblast reporter lineages. This study lays the groundwork for future efficient in vivo CF tracking and functional analyses.

The use of allogeneic adipose-derived mesenchymal stromal cells (alloADSCs) represents an attractive approach for treating myocardial infarction (MI). Furthermore, adding a natural support improves alloADSCs engraftment and survival in heart tissues, leading to a greater therapeutic effect. We aimed to examine the safety and immunological reaction induced by epicardial implantation of a clinical-grade collagen scaffold (CS) seeded with alloADSCs for its future application in humans. Thus, cellularized scaffolds were myocardially or subcutaneously implanted in immunosuppressed rodent models. The toxicological parameters were not significantly altered, and tumor formation was not found over the short or long term. Furthermore, biodistribution analyses in the infarcted immunocompetent rats displayed cell engraftment in the myocardium but no migration to other organs. The immunogenicity of alloADSC-CS was also evaluated in a preclinical porcine model of chronic MI; no significant humoral or cellular alloreactive responses were found. Moreover, CS cellularized with human ADSCs cocultured with human allogeneic immune cells produced no alloreactive response. Interestingly, alloADSC-CS significantly inhibited lymphocyte responses, confirming its immunomodulatory action. Thus, alloADSC-CS is likely safe and does not elicit any alloreactive immunological response in the host. Moreover, it exerts an immunomodulatory action, which supports its translation to a clinical setting.

Background: Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function., Methods: Collagen1α1-GFP (green fluorescent protein)-positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing., Results: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor-β signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy., Conclusions: We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies CTHRC1 as a novel regulator of the healing scar process and a target for future translational studies.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are a promising cell source for cardiac repair after myocardial infarction (MI) because they offer several advantages such as potential to remuscularize infarcted tissue, integration in the host myocardium, and paracrine therapeutic effects. However, cell delivery issues have limited their potential application in clinical practice, showing poor survival and engraftment after transplantation. In this work, we hypothesized that the combination of hiPSC-CMs with microparticles (MPs) could enhance long-term cell survival and retention in the heart and consequently improve cardiac repair. CMs were obtained by differentiation of hiPSCs by small-molecule manipulation of the Wnt pathway and adhered to biomimetic poly(lactic-co-glycolic acid) MPs covered with collagen and poly(d-lysine). The potential of the system to support cell survival was analyzed in vitro, demonstrating a 1.70-fold and 1.99-fold increase in cell survival after 1 and 4 days, respectively. The efficacy of the system was tested in a mouse MI model. Interestingly, 2 months after administration, transplanted hiPSC-CMs could be detected in the peri-infarct area. These cells not only maintained the cardiac phenotype but also showed in vivo maturation and signs of electrical coupling. Importantly, cardiac function was significantly improved, which could be attributed to a paracrine effect of cells. These findings suggest that MPs represent an excellent platform for cell delivery in the field of cardiac repair, which could also be translated into an enhancement of the potential of cell-based therapies in other medical applications., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Coronary heart disease is the leading cause of death worldwide with huge socio-economic consequences. Cell therapy, and particularly mesenchymal stem cells (MSC), are considered a promising option to treat this disorder, due to their robust trophic and immunomodulatory properties. However, limitations such as their low rate of engraftment and poor survival after administration into the heart have precluded their large-scale clinical use. Nevertheless, the combination of MSC with polymer-made scaffolds or hydrogels has proven to enhance their retention and, therefore, their efficacy. Additionally, their allogeneic use could permit the creation of ready-to-use cell patches able to improve their feasibility and promote their application in clinical settings. In this review, the experimental and clinical results derived from the use of MSC in cardiac pathology, as well as advances in the bioengineering field to improve the potential of therapeutic cells, are extensively discussed. Additionally, the current understanding of the heart response to the allogeneic MSC transplants is addressed.

Objective- Cardiac progenitor cells reside in the heart in adulthood, although their physiological relevance remains unknown. Here, we demonstrate that after myocardial infarction, adult Bmi1 + (B lymphoma Mo-MLV insertion region 1 homolog [PCGF4]) cardiac cells are a key progenitor-like population in cardiac neovascularization during ventricular remodeling. Approach and Results- These cells, which have a strong in vivo differentiation bias, are a mixture of endothelial- and mesenchymal-related cells with in vitro spontaneous endothelial cell differentiation capacity. Genetic lineage tracing analysis showed that heart-resident Bmi1 + progenitor cells proliferate after acute myocardial infarction and differentiate to generate de novo cardiac vasculature. In a mouse model of induced myocardial infarction, genetic ablation of these cells substantially deteriorated both heart angiogenesis and the ejection fraction, resulting in an ischemic-dilated cardiac phenotype. Conclusions- These findings imply that endothelial-related Bmi1 + progenitor cells are necessary for injury-induced neovascularization in adult mouse heart and highlight these cells as a suitable therapeutic target for preventing dysfunctional left ventricular remodeling after injury.

Background: The safety and efficacy of cardiac stem/progenitor cells (CSC) have been demonstrated in previous preclinical and clinical assays for heart failure. However, their optimal delivery route to the ischemic heart has not yet been assessed. This study was designed to determine by a non-invasive imaging technique (PET/CT) the biodistribution and acute retention of allogeneic pig CSC implanted by two different delivery routes, intracoronary (IC) and intramyocardial (IM), in a swine preclinical model of chronic ischemia-reperfusion., Methods: Ischemia-reperfusion was induced in six Goettingen hybrid minipigs by 90 min coronary artery occlusion followed by reperfusion. Thirty days later, animals were allocated to receive IC (n = 3) or NOGA ® -guided IM injection (n = 3) of 50 million of 18 F-FDG/GFP-labeled allogeneic pig CSC. Acute retention was quantified by PET/CT 4 h after injection and cell engraftment assessed by immunohistochemical quantification of GFP + cells three days post-injection., Results: Biodistribution of 18 F-FDG-labeled CSC was clearly visualized by PET/CT imaging and quantified. No statistical differences in acute cell retention (percentage of injected dose, %ID) were found in the heart when cells were administered by NOGA ® -guided IM (13.4 ± 3.4%ID) or IC injections (17.4 ± 4.1%ID). Interestingly, engrafted CSC were histologically detected only after IM injection., Conclusion: PET/CT imaging of 18 F-FDG-labeled CSC allows quantifying biodistribution and acute retention of implanted cells in a clinically relevant pig model of chronic myocardial infarction. Similar levels of acute retention are achieved when cells are IM or IC administered. However, acute cell retention does not correlate with cell engraftment, which is improved by IM injection.

Cardiovascular protein therapeutics such as neuregulin (NRG1) and acidic-fibroblast growth factor (FGF1) requires new formulation strategies that allow for sustained bioavailability of the drug in the infarcted myocardium. However, there is no FDA-approved injectable protein delivery platform due to translational concerns about biomaterial administration through cardiac catheters. We therefore sought to evaluate the efficacy of percutaneous intramyocardial injection of poly(lactic-co-glycolic acid) microparticles (MPs) loaded with NRG1 and FGF1 using the NOGA MYOSTAR injection catheter in a porcine model of ischemia-reperfusion. NRG1- and FGF1-loaded MPs were prepared using a multiple emulsion solvent-evaporation technique. Infarcted pigs were treated one week after ischemia-reperfusion with MPs containing NRG1, FGF1 or non-loaded MPs delivered via clinically-translatable percutaneous transendocardial-injection. Three months post-treatment, echocardiography indicated a significant improvement in systolic and diastolic cardiac function. Moreover, improvement in bipolar voltage and decrease in transmural infarct progression was demonstrated by electromechanical NOGA-mapping. Functional benefit was associated with an increase in myocardial vascularization and remodeling. These findings in a large animal model of ischemia-reperfusion demonstrate the feasibility and efficacy of using MPs as a delivery system for growth factors and provide strong evidence to move forward with clinical studies using therapeutic proteins combined with catheter-compatible biomaterials.

Background: Blood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models, but outgrowth characteristics and functionality of BOECs from patients with ischemic cardiomyopathy (ICMP) are unknown. We compared outgrowth efficiency and in vitro and in vivo functionality of BOECs derived from ICMP with BOECs from age-matched (ACON) and healthy young (CON) controls., Methods and Results: We isolated 3.6±0.6 BOEC colonies/100×10(6) mononuclear cells (MNCs) from 60-mL blood samples of ICMP patients (n=45; age: 66±1 years; LVEF: 31±2%) versus 3.5±0.9 colonies/100×10(6) MNCs in ACON (n=32; age: 60±1 years) and 2.6±0.4 colonies/100×10(6) MNCs in CON (n=55; age: 34±1 years), P=0.29. Endothelial lineage (VEGFR2(+)/CD31(+)/CD146(+)) and progenitor (CD34(+)/CD133(-)) marker expression was comparable in ICMP and CON. Growth kinetics were similar between groups (P=0.38) and not affected by left ventricular systolic dysfunction, maladaptive remodeling, or presence of cardiovascular risk factors in ICMP patients. In vitro neovascularization potential, assessed by network remodeling on Matrigel and three-dimensional spheroid sprouting, did not differ in ICMP from (A)CON. Secretome analysis showed a marked proangiogenic profile, with highest release of angiopoietin-2 (1.4±0.3×10(5) pg/10(6) ICMP-BOECs) and placental growth factor (5.8±1.5×10(3) pg/10(6) ICMP BOECs), independent of age or ischemic disease. Senescence-associated β-galactosidase staining showed comparable senescence in BOECs from ICMP (5.8±2.1%; n=17), ACON (3.9±1.1%; n=7), and CON (9.0±2.8%; n=13), P=0.19. High-resolution microcomputed tomography analysis in the ischemic hindlimb of nude mice confirmed increased arteriogenesis in the thigh region after intramuscular injections of BOECs from ICMP (P=0.025; n=8) and CON (P=0.048; n=5) over vehicle control (n=8), both to a similar extent (P=0.831)., Conclusions: BOECs can be successfully culture-expanded from patients with ICMP. In contrast to impaired functionality of ICMP-derived bone marrow MNCs, BOECs retain a robust proangiogenic profile, both in vitro and in vivo, with therapeutic potential for targeting ischemic disease., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Although efforts continue to find new therapies to regenerate infarcted heart tissue, knowledge of the cellular and molecular mechanisms involved remains poor., Objectives: This study sought to identify the origin of cardiac fibroblasts (CFs) in the infarcted heart to better understand the pathophysiology of ventricular remodeling following myocardial infarction (MI)., Methods: Permanent genetic tracing of epicardium-derived cell (EPDC) and bone marrow-derived blood cell (BMC) lineages was established using Cre/LoxP technology. In vivo gene and protein expression studies, as well as in vitro cell culture assays, were developed to characterize EPDC and BMC interaction and properties., Results: EPDCs, which colonize the cardiac interstitium during embryogenesis, massively differentiate into CFs after MI. This response is disease-specific, because angiotensin II-induced pressure overload does not trigger significant EPDC fibroblastic differentiation. The expansion of epicardial-derived CFs follows BMC infiltration into the infarct site; the number of EPDCs equals that of BMCs 1 week post-infarction. BMC-EPDC interaction leads to cell polarization, packing, massive collagen deposition, and scar formation. Moreover, epicardium-derived CFs display stromal properties with respect to BMCs, contributing to the sustained recruitment of circulating cells to the damaged zone and the cardiac persistence of hematopoietic progenitors/stem cells after MI., Conclusions: EPDCs, but not BMCs, are the main origin of CFs in the ischemic heart. Adult resident EPDC contribution to the CF compartment is time- and disease-dependent. Our findings are relevant to the understanding of post-MI ventricular remodeling and may contribute to the development of new therapies to treat this disease., (Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Infarcted hearts are macroscopically stiffer than healthy organs. Nevertheless, although cell behavior is mediated by the physical features of the cell niche, the intrinsic micromechanical properties of healthy and infarcted heart extracellular matrix (ECM) remain poorly characterized. Using atomic force microscopy, we studied ECM micromechanics of different histological regions of the left ventricle wall of healthy and infarcted mice. Hearts excised from healthy (n=8) and infarcted mice (n=8) were decellularized with sodium dodecyl sulfate and cut into 12 μm thick slices. Healthy ventricular ECM revealed marked mechanical heterogeneity across histological regions of the ventricular wall with the effective Young's modulus ranging from 30.2 ± 2.8 to 74.5 ± 8.7 kPa in collagen- and elastin-rich regions of the myocardium, respectively. Infarcted ECM showed a predominant collagen composition and was 3-fold stiffer than collagen-rich regions of the healthy myocardium. ECM of both healthy and infarcted hearts exhibited a solid-like viscoelastic behavior that conforms to two power-law rheology. Knowledge of intrinsic micromechanical properties of the ECM at the length scale at which cells sense their environment will provide further insight into the cell-scaffold interplay in healthy and infarcted hearts., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Although transplantation of adipose-derived stem cells (ADSC) in chronic myocardial infarction (MI) models is associated with functional improvement, its therapeutic value is limited due to poor long-term cell engraftment and survival. Thus, the objective of this study was to examine whether transplantation of collagen patches seeded with ADSC could enhance cell engraftment and improve cardiac function in models of chronic MI. With that purpose, chronically infarcted Sprague-Dawley rats (n = 58) were divided into four groups and transplanted with media, collagen scaffold (CS), rat ADSC, or CS seeded with rat ADSC (CS-rADSC). Cell engraftment, histological changes, and cardiac function were assessed 4 months after transplantation. In addition, Göttingen minipigs (n = 18) were subjected to MI and then transplanted 2 months later with CS or CS seeded with autologous minipig ADSC (CS-pADSC). Functional and histological assessments were performed 3 months post-transplantation. Transplantation of CS-rADSC was associated with increased cell engraftment, significant improvement in cardiac function, myocardial remodeling, and revascularization. Moreover, transplantation of CS-pADSC in the pre-clinical swine model improved cardiac function and was associated with decreased fibrosis and increased vasculogenesis. In summary, transplantation of CS-ADSC resulted in enhanced cell engraftment and was associated with a significant improvement in cardiac function and myocardial remodeling., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The use of scaffolds composed of natural biodegradable matrices represents an attractive strategy to circumvent the lack of cell engraftment, a major limitation of stem cell therapy in cardiovascular diseases. Bovine-derived non-porous collagen scaffolds with different degrees of cross-linking (C0, C2, C5 and C10) were produced and tested for their mechanical behavior, in vitro biocompatibility with adipose-derived stem cells (ADSCs) and tissue adhesion and inflammatory reaction. Uniaxial tensile tests revealed an anisotropic behavior of collagen scaffolds (2×0.5cm) and statistically significant differences in the mechanical behavior between cross-linked and non-cross-linked scaffolds (n=5). In vitro, ADSCs adhered homogenously and showed a similar degree of proliferation on all four types of scaffolds (cells×10(3)cm(-2) at day 7: C0: 94.7±37.1; C2: 91.7±25.6; C5: 88.2±6.8; C10: 72.8±10.7; P=n.s.; n=3). In order to test the in vivo biocompatibility, a chronic myocardial infarction model was performed in rats and 1.2×1.2cm size collagen scaffolds implanted onto the heart 1month post-infarction. Six animals per group were killed 2, 7 and 30days after transplant. Complete and long-lasting adhesion to the heart was only observed with the non-cross-linked scaffolds with almost total degradation 1month post-transplantation. After 7 and 30days post-implantation, the degree of inflammation was significantly lower in the hearts treated with non-cross-linked scaffolds (day 7: C0: 10.2±2.1%; C2: 16.3±2.9%; C5: 15.9±4.8%; C10: 17.4±4.1%; P<0.05 vs. C0; day 30: C0: 1.3±1.3%; C2: 9.4±3.0%; C5: 7.0±2.1%; C10: 9.8±2.5%; P<0.01 vs. C0). In view of the results, the non-cross-linked scaffold (C0) was chosen as an ADSC-carrier sheet and tested in vivo. One week post-implantation, 25.3±7.0% of the cells transplanted were detected in those animals receiving the cell-carrier sheet whereas no cells were found in animals receiving cells alone (n=3 animals/group). We conclude that the biocompatibility and mechanical properties of the non-cross-linked collagen scaffolds make them a useful cell carrier that greatly favors tissue cell engraftment and may be exploited for cell transplantation in models of cardiac disease., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: The aim of this article is to present an optimized acquisition and analysis protocol for the echocardiographic evaluation of left ventricle (LV) remodeling in a mouse model of myocardial infarction (MI)., Methodology: 13 female DBA/2J mice underwent permanent occlusion of the left anterior descending (LAD) coronary artery leading to MI. Mice echocardiography was performed using a Vevo 770 (Visualsonics, Canada) before infarction, and 7, 14, 30, 60, 90 and 120 days after LAD ligation. LV systolic function was evaluated using different parameters, including the fractional area change (FAC%) computed in four high-temporal resolution B-mode short axis images taken at different ventricular levels, and in one parasternal long axis. Pulsed wave and tissue Doppler modes were used to evaluate the diastolic function and Tei Index for global cardiac function. The echocardiographic measurements of infarct size were validated histologically using collagen deposition labeled by Sirius red staining. All data was analyzed using Shapiro-Wilk and Student's t-tests., Principal Findings: Our results reveal LV dilation resulting in marked remodeling an severe systolic dysfunction, starting seven days after MI (LV internal apical diameter, basal = 2.82±0.24, 7d = 3.49±0.42; p<0.001. End-diastolic area, basal = 18.98±1.81, 7d = 22.04±2.11; p<0.001). A strong statistically significant negative correlation exists between the infarct size and long-axis FAC% (r = -0.946; R(2) = 0.90; p<0.05). Moreover, the measured Tei Index values confirmed significant post-infarction impairment of the global cardiac function (basal = 0.46±0.07, 7d = 0.55±0.08, 14 d = 0.57±0.06, 30 d = 0.54±0.06, 60 d = 0.54±0.07, 90 d = 0.57±0.08; p<0.01)., Conclusions/significance: In summary, we have performed a complete characterization of LV post-infarction remodeling in a DBA/2J mouse model of MI, using parameters adapted to the particular characteristics of the model In the future, this well characterized model will be used in both investigative and pharmacological studies that require accurate quantitative monitoring of cardiac recovery after myocardial infarction.

In recent years, the incredible boost in stem cell research has kindled the expectations of both patients and physicians. Mesenchymal progenitors, owing to their availability, ease of manipulation, and therapeutic potential, have become one of the most attractive options for the treatment of a wide range of diseases, from cartilage defects to cardiac disorders. Moreover, their immunomodulatory capacity has opened up their allogenic use, consequently broadening the possibilities for their application. In this review, we will focus on their use in the therapy of myocardial infarction, looking at their characteristics, in vitro and in vivo mechanisms of action, as well as clinical trials.

Since the discovery of the Vascular Endothelial Growth Factor (VEGF) and its leading role in the angiogenic process, this has been seen as a promising molecule for promoting neovascularization in the infarcted heart. However, even though several clinical trials were initiated, no therapeutic effects were observed, due in part to the short half life of this factor when administered directly to the tissue. In this context, drug delivery systems appear to offer a promising strategy to overcome limitations in clinical trials of VEGF.The aim of this paper is to review the principal drug delivery systems that have been developed to administer VEGF in cardiovascular disease. Studies published in the last 5 years are reviewed and the main features of these systems are explained. The tissue engineering concept is introduced as a therapeutic alternative that holds promise for the near future.

Aims: Although transplantation of skeletal myoblast (SkM) in models of chronic myocardial infarction (MI) induces an improvement in cardiac function, the limited engraftment remains a major limitation. We analyse in a pre-clinical model whether the sequential transplantation of autologous SkM by percutaneous delivery was associated with increased cell engraftment and functional benefit., Methods and Results: Chronically infarcted Goettingen minipigs (n = 20) were divided in four groups that received either media control or one, two, or three doses of SkM (mean of 329.6 x 10(6) cells per dose) at intervals of 6 weeks and were followed for a total of 7 months. At the time of sacrifice, cardiac function was significantly better in animals treated with SkM in comparison with the control group. A significantly greater increase in the DeltaLVEF was detected in animals that received three doses vs. a single dose of SkM. A correlation between the total number of transplanted cells and the improvement in LVEF and DeltaLVEF was found (P < 0.05). Skeletal myoblast transplant was associated with an increase in tissue vasculogenesis and decreased fibrosis (collagen vascular fraction) and these effects were greater in animals receiving three doses of cells., Conclusion: Repeated injection of SkM in a model of chronic MI is feasible and safe and induces a significant improvement in cardiac function.

Background: The discrepancy between the functional improvements yielded experimentally by skeletal myoblasts (SM) transplanted in infarcted myocardium and the paucity of their long-term engraftment has raised the hypothesis of cell-mediated paracrine mechanisms., Methods and Results: We analyzed gene expression and growth factors released by undifferentiated human SM (CD56(+)), myotubes (SM cultured until confluence) and fibroblasts-like cells (CD56(-)). Gene expression revealed up-regulation of pro-angiogenic (PGF), anti-apoptotics (BAG-1, BCL-2), heart development (TNNT2, TNNC1) and extracellular matrix remodelling (MMP-2, MMP-7) genes in SM. In line with the gene expression profile, the analysis of culture supernatants of SM by ELISA identified the release of growth factors involved in angiogenesis (VEGF, PIGF, angiogenin, angiopoietin, HGF and PDGF-BB) as well as proteases involved in matrix remodelling (MMP2, MMP9 and MMP10) and their inhibitors (TIMPs). Culture of smooth muscle cells (SMC), cardiomyocytes (HL-1) and human umbilical vein endothelial cells (HUVECs) with SM-released conditioned media demonstrated an increased proliferation of HUVEC, SMC and cardiomyocytes (p<0.05) and a decrease in apoptosis of cardiomyocytes (p<0.05). Analysis of nude rats transplanted with human SM demonstrated expression of human-specific MMP-2, TNNI3, CNN3, PGF, TNNT2, PAX7, TGF-beta, and IGF-1 1 month after transplant., Conclusions: Our data support the paracrine hypothesis whereby myoblast-secreted factors may contribute to the beneficial effects of myogenic cell transplantation in infarcted myocardium.