HDACi vorinostat protects muscle from degeneration after acute rotator cuff injury in mice.

Aims: Rotator cuff (RC) injuries are characterized by tendon rupture, muscle atrophy, retraction, and fatty infiltration, which increase injury severity and jeopardize adequate tendon repair. Epigenetic drugs, such as histone deacetylase inhibitors (HDACis), possess the capacity to redefine the molecular signature of cells, and they may have the potential to inhibit the transformation of the fibro-adipogenic progenitors (FAPs) within the skeletal muscle into adipocyte-like cells, concurrently enhancing the myogenic potential of the satellite cells. Methods: HDACis were added to FAPs and satellite cell cultures isolated from mice. The HDACi vorinostat was additionally administered into a RC injury animal model. Histological analysis was carried out on the isolated supra- and infraspinatus muscles to assess vorinostat anti-muscle degeneration potential. Results: Vorinostat, a HDACi compound, blocked the adipogenic transformation of muscle-associated FAPs in culture, promoting myogenic progression of the satellite cells. Furthermore, it protected muscle from degeneration after acute RC in mice in the earlier muscle degenerative stage after tenotomy. Conclusion: The HDACi vorinostat may be a candidate to prevent early muscular degeneration after RC injury.

Generation of heart and vascular system in rodents by blastocyst complementation.

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.

Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-β and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and β-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action. (AU)

Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia.

Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

An engineered periosteum for efficient delivery of rhBMP-2 and mesenchymal progenitor cells during bone regeneration.

During bone regeneration, the periosteum acts as a carrier for key regenerative cues, delivering osteochondroprogenitor cells and crucial growth factors to the injured bone. We developed a biocompatible, 3D polycaprolactone (PCL) melt electro-written membrane to act as a mimetic periosteum. Poly (ethyl acrylate) coating of the PCL membrane allowed functionalization, mediated by fibronectin and low dose recombinant human BMP-2 (rhBMP-2) (10-25 µg/ml), resulting in efficient, sustained osteoinduction in vitro. In vivo, rhBMP-2 functionalized mimetic periosteum demonstrated regenerative potential in the treatment of rat critical-size femoral defects with highly efficient healing and functional recovery (80%-93%). Mimetic periosteum has also proven to be efficient for cell delivery, as observed through the migration of transplanted periosteum-derived mesenchymal cells to the bone defect and their survival. Ultimately, mimetic periosteum demonstrated its ability to deliver key stem cells and morphogens to an injured site, exposing a therapeutic and translational potential in vivo when combined with unprecedentedly low rhBMP-2 doses.

Cardiac Progenitor Cell Exosomal miR-935 Protects against Oxidative Stress.

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-miRSEL). 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-miRSEL 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-miRSEL, 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.

Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-ß and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and ß-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action.

A Multimodal Scaffold for SDF1 Delivery Improves Cardiac Function in a Rat Subacute Myocardial Infarct Model.

Ischemic heart disease is one of the leading causes of death worldwide. The efficient delivery of therapeutic growth factors could counteract the adverse prognosis of post-myocardial infarction (post-MI). In this study, a collagen hydrogel that is able to load and appropriately deliver pro-angiogenic stromal cell-derived factor 1 (SDF1) was physically coupled with a compact collagen membrane in order to provide the suture strength required for surgical implantation. This bilayer collagen-on-collagen scaffold (bCS) showed the suitable physicochemical properties that are needed for efficient implantation, and the scaffold was able to deliver therapeutic growth factors after MI. In vitro collagen matrix biodegradation led to a sustained SDF1 release and a lack of cytotoxicity in the relevant cell cultures. In vivo intervention in a rat subacute MI model resulted in the full integration of the scaffold into the heart after implantation and biocompatibility with the tissue, with a prevalence of anti-inflammatory and pro-angiogenic macrophages, as well as evidence of revascularization and improved cardiac function after 60 days. Moreover, the beneficial effect of the released SDF1 on heart remodeling was confirmed by a significant reduction in cardiac tissue stiffness. Our findings demonstrate that this multimodal scaffold is a desirable matrix that can be used as a drug delivery system and a scaffolding material to promote functional recovery after MI.

Loss of the matrix metalloproteinase-10 causes premature features of aging in satellite cells.

Aged muscles accumulate satellite cells with a striking decline response to damage. Although intrinsic defects in satellite cells themselves are the major contributors to aging-associated stem cell dysfunction, increasing evidence suggests that changes in the muscle-stem cell local microenvironment also contribute to aging. Here, we demonstrate that loss of the matrix metalloproteinase-10 (MMP-10) in young mice alters the composition of the muscle extracellular matrix (ECM), and specifically disrupts the extracellular matrix of the satellite cell niche. This situation causes premature features of aging in the satellite cells, contributing to their functional decline and a predisposition to enter senescence under proliferative pressure. Similarly, reduction of MMP-10 levels in young satellite cells from wild type animals induces a senescence response, while addition of the protease delays this program. Significantly, the effect of MMP-10 on satellite cell aging can be extended to another context of muscle wasting, muscular dystrophy. Systemic treatment of mdx dystrophic mice with MMP-10 prevents the muscle deterioration phenotype and reduces cellular damage in the satellite cells, which are normally under replicative pressure. Most importantly, MMP-10 conserves its protective effect in the satellite cell-derived myoblasts isolated from a Duchenne muscular dystrophy patient by decreasing the accumulation of damaged DNA. Hence, MMP-10 provides a previously unrecognized therapeutic opportunity to delay satellite cell aging and overcome satellite cell dysfunction in dystrophic muscles.

Revealing cell populations catching the early stages of human embryo development in naive pluripotent stem cell cultures.

Naive human pluripotent stem cells (hPSCs) are defined as the in vitro counterpart of the human preimplantation embryo's epiblast and are used as a model system to study developmental processes. In this study, we report the discovery and characterization of distinct cell populations coexisting with epiblast-like cells in 5iLAF naive human induced PSC (hiPSC) cultures. It is noteworthy that these populations closely resemble different cell types of the human embryo at early developmental stages. While epiblast-like cells represent the main cell population, interestingly we detect a cell population with gene and transposable element expression profile closely resembling the totipotent eight-cell (8C)-stage human embryo, and three cell populations analogous to trophectoderm cells at different stages of their maturation process: transition, early, and mature stages. Moreover, we reveal the presence of cells resembling primitive endoderm. Thus, 5iLAF naive hiPSC cultures provide an excellent opportunity to model the earliest events of human embryogenesis, from the 8C stage to the peri-implantation period.

Development of an injectable alginate-collagen hydrogel for cardiac delivery of extracellular vesicles.

Extracellular vesicles (EVs) are nanosized particles with attractive therapeutic potential for cardiac repair. However, low retention and stability after systemic administration limit their clinical translation. As an alternative, the combination of EVs with biomaterial-based hydrogels (HGs) is being investigated to increase their exposure in the myocardium and achieve an optimal therapeutic effect. In this study, we developed and characterized a novel injectable in-situ forming HG based on alginate and collagen as a cardiac delivery vehicle for EVs. Different concentrations of alginate and collagen crosslinked with calcium gluconate were tested. Based on injectability studies, 1% alginate, 0.5 mg/mL collagen and 0.25% calcium gluconate HG was selected as the idoneous combination for cardiac administration using catheter-based systems. Rheological examination revealed that the HG possessed an internal gel structure, weak mechanical properties and low viscosity, facilitating an easy administration. In addition, EVs were successfully incorporated and homogeneously distributed in the HG. After administration in a rat model of myocardial infarction, the HG showed long-term retention in the heart and allowed for a sustained release of EVs for at least 7 days. Thus, the combination of HGs and EVs represents a promising therapeutic strategy for myocardial repair. Besides EVs delivery, the developed HG could represent a useful platform for cardiac delivery of multiple therapeutic agents.

Comparative Evaluation of Inducible Cre Mouse Models for Fibroblast Targeting in the Healthy and Infarcted Myocardium.

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 (Pdgfra-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 PDGFRa-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.

One-Step In Vitro Generation of ETV2-Null Pig Embryos.

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.

Deficiency of MMP-10 Aggravates the Diseased Phenotype of Aged Dystrophic Mice.

Matrix metalloproteinases (MMPs) have been implicated in the progression of muscular dystrophy, and recent studies have reported the role of MMP-10 in skeletal muscle pathology of young dystrophic mice. Nevertheless, its involvement in dystrophin-deficient hearts remains unexplored. Here, we aimed to investigate the involvement of MMP-10 in the progression of severe muscular dystrophy and to characterize MMP-10 loss in skeletal and cardiac muscles of aged dystrophic mice. We examined the histopathological effect of MMP-10 ablation in aged mdx mice, both in the hind limb muscles and heart tissues. We found that MMP-10 loss compromises survival rates of aged mdx mice, with skeletal and cardiac muscles developing a chronic inflammatory response. Our findings indicate that MMP-10 is implicated in severe muscular dystrophy progression, thus identifying a new area of research that could lead to future therapies for dystrophic muscles.

Local Preirradiation of Infarcted Cardiac Tissue Substantially Enhances Cell Engraftment.

The success of cell therapy for the treatment of myocardial infarction depends on finding novel approaches that can substantially implement the engraftment of the transplanted cells. In order to enhance cell engraftment, most studies have focused on the pretreatment of transplantable cells. Here we have considered an alternative approach that involves the preconditioning of infarcted heart tissue to reduce endogenous cell activity and thus provide an advantage to our exogenous cells. This treatment is routinely used in other tissues such as bone marrow and skeletal muscle to improve cell engraftment, but it has never been taken in cardiac tissue. To avoid long-term cardiotoxicity induced by full heart irradiation we developed a rat model of a catheter-based heart irradiation system to locally impact a delimited region of the infarcted cardiac tissue. As proof of concept, we transferred ZsGreen+ iPSCs in the infarcted heart, due to their ease of use and detection. We found a very significant increase in cell engraftment in preirradiated rats. In this study, we demonstrate for the first time that preconditioning the infarcted cardiac tissue with local irradiation can substantially enhance cell engraftment.

Preclinical Evaluation of the Safety and Immunological Action of Allogeneic ADSC-Collagen Scaffolds in the Treatment of Chronic Ischemic Cardiomyopathy.

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.

Nebulized ivermectin for COVID-19 and other respiratory diseases, a proof of concept, dose-ranging study in rats.

Ivermectin is a widely used antiparasitic drug with known efficacy against several single-strain RNA viruses. Recent data shows significant reduction of SARS-CoV-2 replication in vitro by ivermectin concentrations not achievable with safe doses orally. Inhaled therapy has been used with success for other antiparasitics. An ethanol-based ivermectin formulation was administered once to 14 rats using a nebulizer capable of delivering particles with alveolar deposition. Rats were randomly assigned into three target dosing groups, lower dose (80-90 mg/kg), higher dose (110-140 mg/kg) or ethanol vehicle only. A toxicology profile including behavioral and weight monitoring, full blood count, biochemistry, necropsy and histological examination of the lungs was conducted. The pharmacokinetic profile of ivermectin in plasma and lungs was determined in all animals. There were no relevant changes in behavior or body weight. There was a delayed elevation in muscle enzymes compatible with rhabdomyolysis, that was also seen in the control group and has been attributed to the ethanol dose which was up to 11 g/kg in some animals. There were no histological anomalies in the lungs of any rat. Male animals received a higher ivermectin dose adjusted by adipose weight and reached higher plasma concentrations than females in the same dosing group (mean Cmax 86.2 ng/ml vs. 26.2 ng/ml in the lower dose group and 152 ng/ml vs. 51.8 ng/ml in the higher dose group). All subjects had detectable ivermectin concentrations in the lungs at seven days post intervention, up to 524.3 ng/g for high-dose male and 27.3 ng/g for low-dose females. nebulized ivermectin can reach pharmacodynamic concentrations in the lung tissue of rats, additional experiments are required to assess the safety of this formulation in larger animals.

Single-Cell RNA Sequencing Analysis Reveals a Crucial Role for CTHRC1 (Collagen Triple Helix Repeat Containing 1) Cardiac Fibroblasts After Myocardial Infarction.

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-ß 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.

Induction of Cyclooxygenase-2 by Overexpression of the Human NADPH Oxidase 5 (NOX5) Gene in Aortic Endothelial Cells.

Oxidative stress is a main molecular mechanism that underlies cardiovascular diseases. A close relationship between reactive oxygen species (ROS) derived from NADPH oxidase (NOX) activity and the prostaglandin (PG) biosynthesis pathway has been described. However, little information is available about the interaction between NOX5 homolog-derived ROS and the PG pathway in the cardiovascular context. Our main goal was to characterize NOX5-derived ROS effects in PG homeostasis and their potential relevance in cardiovascular pathologies. For that purpose, two experimental systems were employed: an adenoviral NOX5-ß overexpression model in immortalized human aortic endothelial cells (TeloHAEC) and a chronic infarction in vivo model developed from a conditional endothelial NOX5 knock-in mouse. NOX5 increased cyclooxygenase-2 isoform (COX-2) expression and prostaglandin E2 (PGE2) production through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in TeloHAEC. Protein kinase C (PKC) activation and intracellular calcium level (Ca++) mobilization increased ROS production and NOX5 overexpression, which promoted a COX-2/PGE2 response in vitro. In the chronic infarction model, mice encoding endothelial NOX5 enhanced the cardiac mRNA expression of COX-2 and PGES, suggesting a COX-2/PGE2 response to NOX5 presence in an ischemic situation. Our data support that NOX5-derived ROS may modulate the COX-2/PGE2 axis in endothelial cells, which might play a relevant role in the pathophysiology of heart infarction.

Effect of heart ischemia and administration route on biodistribution and transduction efficiency of AAV9 vectors.

Adeno-associated viruses (AAV) have become one of the most promising tools for gene transfer in clinics. Among all the serotypes, AAV9 has been described as the most efficient for cardiac transduction. In order to achieve optimal therapeutic delivery in heart disease, we have explored AAV9 transduction efficiency in an infarcted heart using different routes of administration and promoters, including a cardiac-specific one. AAV9 vectors carrying luciferase or green fluorescence protein under the control of the ubiquitous elongation-factor-1-alpha or the cardiac-specific troponin-T (TnT) promoters were administered by intramyocardial or intravenous injection, either in healthy or myocardial-infarcted mice. The transduction efficacy and specificity, the time-course expression, and the safety of each vector were tested. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV-TnT, which was significantly higher after intramyocardial injection regardless of ischemia-induction. On the contrary, high hepatic transgene expression levels were detected with the elongation-factor-1-alpha-promoter, independently of the administration route and heart damage. Moreover, tissue-specific green fluorescence protein expression was found in cardiomyocytes with the TnT vector, whereas minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that myocardial infarction greatly increased the transcriptional activity of AAV genomes. Our findings show that the use of cardiac promoters allows for specific and stable cardiac gene expression, which is optimal and robust when intramyocardially injected. Furthermore, our data indicate that the pathological status of the tissue can alter the transcriptional activity of AAV genomes, an aspect that should be carefully evaluated for clinical applications.