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Decellularized extracellular matrix in the form of patches and locally injected hydrogels has long been used as therapies in animal models of disease. Here we report the safety and feasibility of an intravascularly infused extracellular matrix as a biomaterial for the repair of tissue in animal models of acute myocardial infarction, traumatic brain injury and pulmonary arterial hypertension. The biomaterial consists of decellularized, enzymatically digested and fractionated ventricular myocardium, localizes to injured tissues by binding to leaky microvasculature, and is largely degraded in about 3 d. In rats and pigs with induced acute myocardial infarction followed by intracoronary infusion of the biomaterial, we observed substantially reduced left ventricular volumes and improved wall-motion scores, as well as differential expression of genes associated with tissue repair and inflammation. Delivering pro-healing extracellular matrix by intravascular infusion post injury may provide translational advantages for the healing of inflamed tissues 'from the inside out'.

Newts have the extraordinary ability for regeneration, including the ability to regrow nerve and retinal tissue, and even amputated limbs. In contrast, mammals lack broad regenerative capabilities. While the molecular basis of newts’ regenerative ability is the subject of active study, the underlying paracrine signaling factors remain largely uncharacterized. Extracellular vesicles (EVs) play an important role in cell-to-cell communication via EV cargo-mediated regulation of gene expression patterns within the recipient cells. Here, we report that newt myogenic precursor cells (A1 cells) secrete EVs (A1EVs) that contain messenger RNAs associated with early embryonic development, neuronal differentiation, and cell survival. Exposure of rat primary superior cervical ganglion (SCG) neurons to A1EVs increased neurite outgrowth, facilitated by increases in mitochondrial respiration. Canonical pathway analysis pinpointed activation of NGF/ERK5 signaling in SCG neurons exposed to A1EV, which we validated experimentally. Thus, newt EVs drive mammalian neurite growth and complexity.

BackgroundPulmonary Arterial Hypertension (PAH) is a progressive cardiopulmonary disease and is characterized by occlusive remodeling of pulmonary arterioles and increased pulmonary vascular resistance. With the onset of PAH, the right ventricle (RV) of the heart adapts to the increased afterload pressure by undergoing adaptive hypertrophic remodeling to maintain adequate blood flow. However, for unknown reasons, maladaptive influences ensue, resulting in impaired RV function with progressive decompensation and right heart failure. Using a rodent model of PAH, we evaluated key signaling pathways mediating cardiac muscle protein synthesis in the RV during the adaptive hypertrophy phase, with preserved right heart function, and the decompensated maladaptive phase, in which right heart failure (RHF) was clinically present.MethodsMale Sprague-Dawley rats were injected subcutaneously with 60mg/kg Monocrotaline (MCT) and RV function was assessed by echocardiography during PAH disease progression. RV tissue was collected during the adaptive and maladaptive phases of PAH and cell signaling pathways involved in survival, hypertrophy, and autophagy, as well as fibrosis and vascularization, were probed using qPCR, Western blotting and histology. Statistical analysis was performed using ANOVA to compare differences between the independent groups and Student-Newman-Keuls test was used to compare differences within independent groups.ResultsAnalysis of protein and gene expression changes in PAH animals identified three key signaling pathways involved in the shift toward maladaptive right heart failure: i) PI3K/Akt/mTOR; ii) GSK-3; iii) MAPK/ERK, as well as IGF-1 regulation. During adaptive hypertrophy, significant increments of phosphorylated proteins in the three signaling pathways were observed with increases in RV fibrosis and decreased capillarity found. In the maladaptive phase, mTORC1 and its downstream effector p-70S6K were significantly activated, contributing to the decreased LC3-I/II ratio, a marker of autophagy inhibition. Additionally, p27, a cyclin-dependent kinase (CDK) inhibitor, which has been recently implicated in regulating mTOR activity to inhibit autophagy and promote heart failure was significantly downregulated. ConclusionWe propose that autophagy inhibition in conjunction with other maladaptive processes reported in the decompensated RV muscle contributes to the genesis of overt RHF in PAH and that a continuum of changes characterizes the adaptive and maladaptive phases in the RV muscle.

Interferon-gamma (IFN-γ), a proinflammatory cytokine, has been used as an early indicator of multiple infectious diseases or tumors. In order to explore the detection capability of a commonly used anti-IFN-γ aptamer, a simple target induced strand-displacement aptasensing strategy was tested by introducing three different complementary strands and two different signal/quencher pairs. The Texas red/BHQ2-based sensor showed the best affinity constant (Kd) of 21.87 ng mL−1. It was found that the strand-displacement aptasensing strategy was impacted by the complementary position and length of the complementary strands. Additionally, the hybridization chain reaction (HCR) amplification strategy was introduced, yielding a 12-fold improved sensitivity of 0.45 ng mL−1. In order to further explore the sensing platform for spatially localized cytokine detection, the Texas red/BHQ2-based strand-displacement aptasensor was successfully fabricated on the 3D optical fiber surface to achieve a deployable sensing device for monitoring IFN-γ based on the fluorescence spots counting strategy. Finally, the three developed aptasensing strategies (strand-displacement strategy, HCR amplification strategy, 3D optical fiber aptasensor) were applied for detection of IFN-γ secreted by PBMCs with comparable results to those of ELISA. The deployable 3D optical fiber aptasensor with the superior sensitivity is potential to be used for detection of spatially localized IFN-γ in vivo.

Biomaterials, such as extracellular matrix (ECM) hydrogels, have been widely used in preclinical studies as injectable tissue engineering therapies; however, injectable therapies are limited as they can cause localized trauma or organ perforation. We have developed a new ECM therapy, the low molecular weight fraction derived from decellularized, digested ECM, for intravascular infusion. This new form of ECM can be infused after injury, specifically localize to injured tissues by coating the leaky microvasculature, and promote cell survival and tissue repair. In this study, we show the feasibility and targeting of intravascular ECM infusions using models of acute myocardial infarction (MI), traumatic brain injury, and pulmonary arterial hypertension. Furthermore, safety and efficacy were demonstrated in small and large animal models of acute MI following intracoronary infusion, which included using a clinically-relevant catheter in the large animal model. Functional improvements, specifically reduced left ventricular volumes and improved wall motion scores were observed after ECM infusions post-MI. Genes related to tissue repair and inflammation were differential expressed in response to ECM infusions. This study shows proof-of-concept for a new paradigm of delivering pro-healing ECM biomaterials via intravascular infusion to heal tissue from the inside out.

Background Extracellular vesicles (EVs) and exosomes are nano-sized, membrane-bound vesicles shed by most eukaryotic cells studied to date. EVs play key signaling roles in cellular development, cancer metastasis, immune modulation and tissue regeneration. Attempts to modify exosomes to increase their targeting efficiency to specific tissue types are still in their infancy. Here we describe an EV membrane anchoring platform termed “cloaking” to directly embed tissue-specific antibodies or homing peptides on EV membrane surfaces ex vivo for enhanced vesicle uptake in cells of interest. The cloaking system consists of three components: DMPE phospholipid membrane anchor, polyethylene glycol spacer and a conjugated streptavidin platform molecule, to which any biotinylated molecule can be coupled for EV decoration. Results We demonstrate the utility of membrane surface engineering and biodistribution tracking with this technology along with targeting EVs for enhanced uptake in cardiac fibroblasts, myoblasts and ischemic myocardium using combinations of fluorescent tags, tissue-targeting antibodies and homing peptide surface cloaks. We compare cloaking to a complementary approach, surface display, in which parental cells are engineered to secrete EVs with fusion surface targeting proteins. Conclusions EV targeting can be enhanced both by cloaking and by surface display; the former entails chemical modification of preformed EVs, while the latter requires genetic modification of the parent cells. Reduction to practice of the cloaking approach, using several different EV surface modifications to target distinct cells and tissues, supports the notion of cloaking as a platform technology. Electronic supplementary material The online version of this article (10.1186/s12951-018-0388-4) contains supplementary material, which is available to authorized users.

Aim The aim is to assess the effects of CDCs on heart structure, function, gene expression, and systemic parameters in aged rats. Diastolic dysfunction is characteristic of aged hearts. Cardiosphere-derived cell (CDC) therapy has exhibited several favourable effects on heart structure and function in humans and in preclinical models; however, the effects of CDCs on aging have not been evaluated. Methods and Results We compared intra-cardiac injections of neonatal rat CDCs to vehicle (phosphate-buffered saline, PBS) in 21.8 ± 1.6 month-old rats (mean ± standard deviation; n = 23 total). Ten rats 4.1 ± 1.5 months of age comprised a young reference group. Blood, echocardiographic, haemodynamic and treadmill stress tests were performed at baseline in all animals, and 1 month after treatment in old animals. Histology and the transcriptome were assessed after terminal phenotyping. For in vitro studies, human heart progenitors from older donors, or cardiomyocytes from aged rats were exposed to human CDCs or exosomes secreted by CDCs (CDC-XO) from paediatric donors. Transcriptomic analysis revealed that CDCs, but not PBS, recapitulated a youthful pattern of gene expression in the hearts of old animals (85.5% of genes differentially expressed, P

In order to satisfy the need for sensitive detection of Aflatoxin M1 (AFM1), we constructed a simple and signal-on fluorescence aptasensor based on an autocatalytic Exonuclease III (Exo III)-assisted signal amplification strategy. In this sensor, the DNA hybridization on magnetic nanobeads could be triggered by the target AFM1, resulting in the release of a single-stranded DNA to induce an Exo III-assisted signal amplification, in which numerous G-quadruplex structures would be produced and then associated with the fluorescent dye to generate significantly amplified fluorescence signals resulting in the increased sensitivity. Under the optimized conditions, this aptasensor was able to detect AFM1 with a practical detection limit of 9.73 ng kg&minus, 1 in milk samples. Furthermore, the prepared sensor was successfully used for detection of AFM1 in the commercially available milk samples with the recovery percentages ranging from 80.13% to 108.67%. Also, the sensor performance was evaluated by the commercial immunoassay kit with satisfactory results.

Technological advances in the current era of big data have made it easy for the high-speed collection and generation of large volumes of a wide variety of valuable data (which may be of different veracity including precise, imprecise and uncertain data). As rich sources of big data, social networks consist of social entities who are often linked by some interdependency such as ‘following’ relationships. Since these big social networks have been growing, there are real-life situations in which an individual user wants to find those frequently followed groups of social entities so that he can follow the same groups. Discovery of these frequently followed groups can be challenging because the social networks are usually big with lots of social entities. In this paper, we present a social data compression scheme and its associated big social data mining algorithm for the discovery of ‘following’ relationships. Evaluation results show the practicality of our compression scheme and its associated algorithm for mining big social data in a cloud computing environment.

In the current era of big data, advanced technology has led to easy collection or generation of high volumes of a wide variety of valuable data of different veracity. As rich sources of big data, social networks consist of users (or social entities) who are often linked by some interdependency such as ‘following’ relationships. Since these big social networks keep growing at a high velocity, there are situations in which an individual user (or business) wants to find those frequently followed groups of social entities so that he can also follow the same groups. Discovery of these frequently followed groups can be challenging because the social networks are usually big (with lots of users/social entities) but can be sparsely distributed (with most users only know some but not all users/social entities in some portions of a social network). In this paper, we present a social network mining algorithm that uses different compressed models to space-efficiently represent social entities so as to facilitate the discovery of groups of frequently followed social entities from these big but sparsely distributed social networks. Evaluation results show the practicality of our algorithm in efficient mining of ‘following’ patterns from big but sparsely distributed social networks.

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, economical and widely used methods for evaluating gene expression. However, the utility of this method continues to be undermined by a number of challenges including normalization using appropriate reference genes. The need to develop tailored and effective strategies is further underscored by the burgeoning field of extracellular vesicle (EV) biology. EVs contain unique signatures of small RNAs including microRNAs (miRs). In this study we develop and validate a comprehensive strategy for identifying highly stable reference genes in a therapeutically relevant cell type, cardiosphere-derived cells. Data were analysed using the four major approaches for reference gene evaluation: NormFinder, GeNorm, BestKeeper and the Delta Ct method. The weighted geometric mean of all of these methods was obtained for the final ranking. Analysis of RNA sequencing identified miR-101-3p, miR-23a-3p and a previously identified EV reference gene, miR-26a-5p. Analysis of a chip-based method (NanoString) identified miR-23a, miR-217 and miR-379 as stable candidates. RT-qPCR validation revealed that the mean of miR-23a-3p, miR-101-3p and miR-26a-5p was the most stable normalization strategy. Here, we demonstrate that a comprehensive approach of a diverse data set of conditions using multiple algorithms reliably identifies stable reference genes which will increase the utility of gene expression evaluation of therapeutically relevant EVs.

Pulmonary arterial hypertension (PAH) is a progressive condition characterized by occlusive pulmonary arteriopathy, in which survival remains poor despite pharmacologic advances. The aim of this study was to evaluate the ability of cardiosphere-derived cells (CDCs), cardiac progenitor cells with potent anti-inflammatory and immunomodulatory properties, to attenuate hemodynamic and morphometric remodeling of the right ventricle (RV) and pulmonary arterioles in rats with established monocrotaline (MCT)-induced PAH. Animals were divided into 3 groups: 1) Control (CTL), 2) PAH in which CDCs were centrally infused (CDC) and 3) PAH in which saline was given (Sham). Significant increments in RV systolic pressure (RVSP) and RV hypertrophy were noted in Sham animals compared to CTL. In CDC rats at day 35, RSVP fell (- 38%; p< 0.001) and RV hypertrophy decreased (-26%; p< 0.01). TAPSE and cardiac output were preserved in all 3 groups at day 35. Pulmonary arteriolar wall thickness was greater in Sham rats compared to CTL, and reduced in CDC animals for vessels 20-50 μm (P

Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non-myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere-derived cells amplifies innate cardioblast-mediated tissue regeneration, in part through the secretion of stromal cell-derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium.

The small heat shock protein HspB1 (Hsp27) is abundantly expressed in embryonic muscle tissues of a wide variety of vertebrate species. However, the functional significance of this expression pattern is not well established. In the present study, we observed specific, high level expression of HspB1 protein and an HspB1 gene reporter in developing craniofacial muscles of the zebrafish, Danio rerio, and examined the consequences of reducing HspB1 expression to the development and growth of these muscles. Quantitative morphometric analyses revealed a reduction in the cross-sectional area of myofibers in embryos expressing reduced HspB1 levels by as much as 47% compared to controls. In contrast, we detected no differences in the number of myofibrils or associated nuclei, nor the number, size or development of chondrocytes in surrounding tissues. We also did not detect changes to the overall organization of sarcomeres or myofibrils in embryos expressing reduced levels of HspB1. Together our results reveal a critical role for HspB1 in the growth of myofibrils and provide new insight into the mechanism underlying its developmental function.

Background Infusion of allogeneic cardiosphere‐derived cells (allo‐ CDC s) postreperfusion elicits cardioprotective cellular postconditioning in pigs with acute myocardial infarction. However, the long‐term effects of allo‐ CDC s have not been assessed. We performed a placebo‐controlled pivotal study for long‐term evaluation, as well as shorter‐term mechanistic studies. Methods and Results Minipigs underwent 1.5‐hour mid‐left anterior descending balloon occlusion followed by reperfusion and were randomized to receive intracoronary allo‐ CDC s or vehicle 30 minutes postreperfusion. Left ventriculography ( LVG ) demonstrated preserved ejection fraction ( EF ) and attenuation of LV remodeling in CDC ‐treated pigs. Pigs underwent cardiac magnetic resonance imaging ( MRI ) and LVG 1 hour and 8 weeks after therapy to evaluate efficacy. MRI showed improvement of EF and attenuation of LV remodeling immediately after allo‐ CDC infusion. In addition, allo‐ CDC s improved regional function and decreased hypertrophy 2 months post‐treatment. Histological analysis revealed increased myocardial salvage index, enhanced vascularity, sustained reductions in infarct size/area at risk and scar transmurality, and attenuation of collagen deposition in the infarct zone of allo‐ CDC ‐treated pigs at 2 months. Allo‐ CDC s did not evoke lymphohistiocytic infiltration or systemic humoral memory response. Short‐term experiments designed to probe mechanism revealed antiapoptotic effects of allo‐ CDC s on cardiomyocytes and increases in cytoprotective macrophages, but no increase in overall inflammatory cell infiltration 2 hours after cell therapy. Conclusions Allo‐ CDC infusion postreperfusion is safe, improves cardiac function, and attenuates scar size and remodeling. The favorable effects persist for at least 2 months after therapy. Thus, cellular postconditioning confers not only acute cardioprotection, but also lasting structural and functional benefits.

Single-vessel, intracoronary infusion of stem cells under stop-flow conditions has proven safe but achieves only limited myocardial coverage. Continuous flow intracoronary delivery to one or more coronary vessels may achieve broader coverage for treating cardiomyopathy, but has not been investigated. Using nonocclusive coronary guiding catheters, we infused allogeneic cardiosphere-derived cells (CDCs) either in a single vessel or sequentially in all three coronary arteries in porcine ischemic cardiomyopathy and used magnetic resonance imaging (MRI) to assess structural and physiological outcomes. Vehicle-infused animals served as controls. Single-vessel stop-flow and continuous-flow intracoronary infusion revealed equivalent effects on scar size and function. Sequential infusion into each of the three major coronary vessels under stop-flow or continuous-flow conditions revealed equal efficacy, but less elevation of necrotic biomarkers with continuous-flow delivery. In addition, multi-vessel delivery resulted in enhanced global and regional tissue function compared to a triple-vessel placebo-treated group. The functional benefits after global cell infusion were accompanied histologically by minimal inflammatory cellular infiltration, attenuated regional fibrosis and enhanced vessel density in the heart. Sequential multi-vessel non-occlusive delivery of CDCs is safe and provides enhanced preservation of left ventricular function and structure. The current findings provide preclinical validation of the delivery method currently undergoing clinical testing in the Dilated cardiomYopathy iNtervention With Allogeneic MyocardIally-regenerative Cells (DYNAMIC) trial of CDCs in heart failure patients.

PurposeThe purpose of this paper is to describe challenges and lessons learned in the design and development of a comprehensive, flexible environmental management system (EMS) in a real university setting; also to inform development of similar systems elsewhere and provide a modular, extensible software architecture for such efforts.Design/methodology/approachA modular, flexible software architecture was designed as the cornerstone of a comprehensive, secure web‐based data collection and analysis framework. Environmental data such as utility usages, waste generation and transportation services were identified, collected, and entered into the evolving system. The system is easily extensible to new environmental data types, and supported manual and automated data entry, custom “at‐the‐source data entry” mechanisms, and flexible tools for visually analyzing environmental data captured.FindingsDevelopment of automated EMS systems for large institutions is significantly complicated by profound heterogeneity in campus infrastructure, management policies, and limited data accessibility; legacy data are often incomplete or inaccurate. Successful EMS initiatives must explicitly address these challenges through realistic project planning, choice of software technologies, design of system architecture, and administrative commitment. Detailed insights in each of the above areas are provided.Originality/valueThe authors provide clarifying discussion of sustainability plans versus monitoring systems, place popular technological gadgets such as live building energy monitors into perspective within this framework, and describe design and implementation of a comprehensive environmental monitoring framework. The modular concept for system architecture, design approach, and lessons learned can inform the development of similar comprehensive EMS development efforts.

Newts can regenerate amputated limbs and cardiac tissue, unlike mammals which lack broad regenerative capacity. Several signaling pathways involved in cell proliferation, differentiation and survival during newt tissue regeneration have been elucidated, however the factors that coordinate signaling between cells, as well as the conservation of these factors in other animals, are not well defined. Here we report that media conditioned by newt limb explant cells (A1 cells) protect mammalian cardiomyocytes from oxidative stress-induced apoptosis. The cytoprotective effect of A1-conditioned media was negated by exposing A1 cells to GW4869, which suppresses the generation of extracellular vesicles (EVs). A1-EVs are similar in diameter (~100–150 nm), structure, and share several membrane surface and cargo proteins with mammalian exosomes. However, isolated A1-EVs contain significantly higher levels of both RNA and protein per particle than mammalian EVs. Additionally, numerous cargo RNAs and proteins are unique to A1-EVs. Of particular note, A1-EVs contain numerous mRNAs encoding nuclear receptors, membrane ligands, as well as transcription factors. Mammalian cardiomyocytes treated with A1-EVs showed increased expression of genes in the PI3K/AKT pathway, a pivotal player in survival signaling. We conclude that newt cells secrete EVs with diverse, distinctive RNA and protein contents. Despite ~300 million years of evolutionary divergence between newts and mammals, newt EVs confer cytoprotective effects on mammalian cardiomyocytes.

Background— Preclinical studies in rodents and pigs indicate that the self-assembling microtissues known as cardiospheres may be more effective than dispersed cardiosphere-derived cells. However, the more desirable intracoronary route has been assumed to be unsafe for cardiosphere delivery: Cardiospheres are large (30–150 μm), raising concerns about likely microembolization. We questioned these negative assumptions by evaluating the safety and efficacy of optimized intracoronary delivery of cardiospheres in a porcine model of convalescent myocardial infarction. Methods and Results— First, we standardized the size of cardiospheres by modifying culture conditions. Then, dosage was determined by infusing escalating doses of cardiospheres in the left anterior descending artery of naive pigs, looking for acute adverse effects. Finally, in a randomized efficacy study, 14 minipigs received allogeneic cardiospheres (1.3×10 6 ) or vehicle 1 month after myocardial infarction. Animals underwent magnetic resonance imaging before infusion and 1 month later to assess left ventricular ejection fraction, scar mass, and viable mass. In the dosing study, we did not observe any evidence of microembolization after cardiosphere infusion. In the post-myocardial infarction study, cardiospheres preserved LV function, reduced scar mass and increased viable mass, whereas placebo did not. Moreover, cardiosphere decreased collagen content, and increased vessel densities and myocardial perfusion. Importantly, intracoronary cardiospheres decreased left ventricular end-diastolic pressure and increased cardiac output. Conclusions— Intracoronary delivery of cardiospheres is safe. Intracoronary cardiospheres are also remarkably effective in decreasing scar, halting adverse remodeling, increasing myocardial perfusion, and improving hemodynamic status after myocardial infarction in pigs. Thus, cardiospheres may be viable therapeutic candidates for intracoronary infusion in selected myocardial disorders.

Background Cardiosphere-derived cells mediate therapeutic regeneration in patients after myocardial infarction and are undergoing further clinical testing for cardiomyopathy. The beneficial effects of cardiosphere-derived cells are mediated by the secretion of exosomes and possibly other extracellular membrane vesicles (EMVs). Objectives This study sought to investigate the effect of cardiosphere-derived EMVs (CSp-EMVs) on fibroblasts in vitro and tested whether priming with CSp-EMVs could confer salutary properties on fibroblasts in vivo. Methods CSp-EMVs were isolated from serum-free media conditioned for 3 days by cardiospheres. Dermal fibroblasts were primed with CSp-EMVs for 24 h followed by exosomal micro-ribonucleic acid profiling. In vivo, we injected CSp-EMV−primed or −unprimed dermal fibroblasts (or CSp-EMVs) in a chronic rat model of myocardial infarction and defined the functional and structural consequences. Results CSp-EMVs amplified their own biological signals: exposure of “inert” fibroblasts to CSp-EMVs rendered the fibroblasts therapeutic. Intramyocardially injected CSp-EMV−primed (but not unprimed) fibroblasts increased global pump function and vessel density while reducing scar mass. CSp-EMV priming caused fibroblasts to secrete much higher levels of stromal-cell-derived factor 1 and vascular endothelial growth factor and dramatically changed the micro-ribonucleic acid profile of fibroblast-secreted EMVs in vitro. The priming was followed by significant angiogenic and cardioprotective effects. Conclusions CSp-EMVs alter fibroblast phenotype and secretome in a salutary positive-feedback loop. The phenotypic conversion of inert cells to therapeutically active cells reveals a novel mechanism for amplification of exosome bioactivity.

Background— Intracoronary delivery of cardiosphere-derived cells (CDCs) has been demonstrated to be safe and effective in porcine and human chronic myocardial infarction. However, intracoronary delivery of CDCs after reperfusion in acute myocardial infarction has never been assessed in a clinically-relevant large animal model. We tested CDCs as adjunctive therapy to reperfusion in a porcine model of myocardial infarction. Methods and Results— First, escalating doses (5, 7.5, and 10 million cells) of allogeneic CDCs were administered intracoronary 30 minutes after reperfusion. Forty-eight hours later, left ventriculography was performed and animals euthanized to measure area at risk, infarct size (IS), and microvascular obstruction. Second, identical end points were measured in a pivotal study of minipigs (n=14) that received 8.5 to 9 million allogeneic CDCs, placebo solution, or sham. Multiple indicators of cardioprotection were observed with 7.5 and 10 million allogeneic CDCs, but not 5 million CDCs, relative to control. In the pivotal study, IS, microvascular obstruction, cardiomyocyte apoptosis, and adverse left ventricular remodeling were all smaller in the CDC group than in sham or placebo groups. In addition, serum troponin I level at 24 hours was lower after CDC infusion than that in the placebo or sham groups, consistent with the histologically-demonstrated reduction in IS. Conclusions— Intracoronary delivery of allogeneic CDCs is safe, feasible, and effective in cardioprotection, reducing IS, preventing microvascular obstruction, and attenuating adverse acute remodeling. This novel cardioprotective effect, which we call cellular postconditioning, differs from previous strategies to reduce IS in that it works even when initiated with significant delay after reflow.

Background: Pre-clinical studies in rodents and pigs indicate that the self-assembling microtissues known as cardiospheres (CSp), when administered intramyocardially, may be more effective than dispersed CSp-derived cells (CDCs). However, the more desirable intracoronary (IC) route has been assumed to be unsafe for CSp delivery: CSp are large (>35 μm), raising concerns about likely microembolization. Objective: We sought to evaluate the safety and efficacy of IC delivery of allogeneic CSp in a porcine model of convalescent MI. Methods: Dosage was optimized by infusing CSp (3.25x10 5 particles [n=2], 6.5 x10 5 [n=3] and 1.3x10 6 [n=2], size=44±23, 29%>50μm) in the LAD of naïve pigs, looking for acute adverse effects (troponin I [TnI] leak, low TIMI flow, stunning). We next tested the efficacy of IC allogeneic Csp (1.3x10 6 ; n=7) or vehicle (n=8) in a minipig model of chronic MI. Animals underwent MRI before infusion and 1 month later. Left ventricular (LV) ejection fraction (EF), scar mass and viable mass were evaluated at both time points. Results: In the dosing study, we observed no impairment of TIMI flow or LVEF after CSp infusion. TnI at 24 hours was 0.7±0.5ng/mL and did not differ among groups (P=0.11). In the post-MI study, EF was identical in the two groups at baseline. One month post-infusion, LV function was preserved in the CSp group but not in controls (ΔEF=+0.5±1.6% vs. -4.5±1.8%, p Conclusions: IC delivery of allogeneic CSp is safe and preserves LV function after MI. In addition, global hemodynamic improvement is observed, which may have significant clinical implications. The decision to use CDCs or CSp is not forced, therefore, by an inability to infuse CSp safely via the IC route.

BACKGROUND: Intracoronary (IC) infusion of cardiosphere-derived stem cells (CDCs) under stop-flow conditions has proven efficacious in producing myocardial regeneration in preclinical and clinical studies. However, the minimally-effective dose is not known, nor is it clear whether benefit is straightforwardly dependent on CDC dose. OBJECTIVE: To determine the dose-efficacy relationship of allogeneic CDCs in a large-animal chronic myocardial infarction (MI) model. METHODS: Escalating doses of allogeneic CDCs (3.125M, 6.25M, 12.5M, 25M; or vehicle as a control) were infused into the left anterior descending coronary artery using a nonocclusive guiding catheter in an anterior chronic MI minipig model. Magnetic resonance imaging (MRI) was used to assess structural and functional endpoints. RESULTS: CDCs were infused in a total of 25 animals under continuous flow at a rate of 1.25M CDCs/min. An additional 5 control animals underwent continuous flow of vehicle over 10 min. No animals experienced adverse events during or after the infusions. One month post therapy, ejection fraction (EF) was preserved at doses of 6.25M, 12.5M and 25M, but not with 3.125 M allo-CDCs or control (ΔEF = -6.7±0.9% in the placebo, -5.1±1.8% in 3.125M, -1.4±0.6% in the 6.25M, -2±1.4% in the 12.5M and -2.3±1.2% in the 25M group; p=0.04 by one way ANOVA for treatment effect). All doses of CDCs (except 3.125M) led to equivalent scar mass reductions, as assessed by MRI with Gd enhancement (p=0.0037 treated vs placebo, one-way ANOVA). Provocative electrophysiology testing at the end of the study revealed no enhanced arrythmogenic potential after cell therapy. CONCLUSIONS: Allogeneic CDCs can be safely administered with a non-occlusive method, over a wide dosage range. All doses >3.125M lead to enhanced preservation of left ventricular function and tissue remodeling, but to similar degrees. Our data reveal that CDCs work by a “threshold” effect that does not obey classical drug-receptor kinetics. This finding is consistent with the extraordinary ability of seemingly-homeopathic doses to exert large, lasting benefits.

Background: Multicellular self-assembling cardiospheres (CSps) exert regenerative and anti-fibrotic effects via paracrine mechanisms. CSp-derived cells are known to secrete exosomes which mediate most or all of the beneficial therapeutic effects. Objective: We evaluated the regenerative capacity of CSp-secreted exosomes in a model of chronic myocardial infarction (MI). We also determined whether CSp-exosomes could convert the phenotype of therapeutically-inert cells. Methods: Exosomes were isolated from CSp-conditioned media by adding a precipitation solution followed by centrifugation. One month post-MI, Wistar Kyoto rats (n=46) with permanent LAD ligation were injected intramyocardially with: a) human dermal fibroblasts (DFs), b) CSp exosomes, c) DFs primed with CSp-exosomes, d) CSps only or e) vehicle. Functional and histological analyses were performed 4 weeks after therapy. Mechanisms were also probed in vitro . Results: In vivo, CSp-exosomes and CSps equally increased ejection fraction (EF= 45±1% [CSp-exo], 44±2% [CSps], 33±1% [placebo] and 35±2% [DFs]) and reduced scar mass (48±8mg [CSp-exo], 45±4mg [CSps], 96±12mg [placebo] and 90±6mg [DFS]; both pIn vitro , exosome-primed DFs increased tube formation by HUVECs and inhibited cardiomyocyte apoptosis. Immunohistochemistry showed increased vessel density in all groups compared to vehicle or unprimed DFs. Conclusions: Administration of CSp-exosomes recapitulates the regenerative potential and functional benefits of CSps themselves. The surprising ability of CSp-exosomes to confer therapeutic efficacy on inert DFs may represent an unanticipated amplification mechanism for exosome-mediated benefits.

Background The regenerative potential of cardiosphere‐derived cells ( CDC s) for ischemic heart disease has been demonstrated in mice, rats, pigs, and a recently completed clinical trial ( CADUCEUS ). CDC s are CD 105 + stromal cells of intrinsic cardiac origin, but the antigenic characteristics of the active fraction remain to be defined. CDC s contain a small minority of c‐kit + cells, which have been argued to be cardiac progenitors, and a variable fraction of CD 90 + cells whose bioactivity is unclear. Methods We performed a retrospective analysis of data from the CADUCEUS trial and a prospective mouse study to elucidate the roles of c‐kit + and CD 90 + cells in human CDC s. Here, we show, surprisingly, that c‐kit expression has no relationship to CDC s' therapeutic efficacy in humans, and depletion of c‐kit + cells does not undermine the structural and functional benefits of CDC s in a mouse model of myocardial infarction (MI). In contrast, CD 90 expression negatively correlates with the therapeutic benefit of CDC s in humans (ie, higher CD 90 expression associated with lower efficacy). Depletion of CD 90 + cells augments the functional potency of CDC s in murine MI. CD 90 − CDC s secrete lower levels of inflammatory cytokines and can differentiate into cardiomyocytes in vitro and in vivo. Conclusion The majority population of CDC s ( CD 105 + / CD 90 − /c‐kit − ) constitutes the active fraction, both in terms of therapeutic efficacy and in the ability to undergo cardiomyogenic differentiation. The c‐kit + fraction is neither necessary for, nor contributory to, the regenerative efficacy of CDC s.

Stem cell transplantation is a promising strategy for therapeutic cardiac regeneration, but current therapies are limited by inefficient interaction between potentially beneficial cells (either exogenously transplanted or endogenously recruited) and the injured tissue. Here we apply targeted nanomedicine to achieve in vivo cell-mediated tissue repair, imaging and localized enrichment without cellular transplantation. Iron nanoparticles are conjugated with two types of antibodies (one against antigens on therapeutic cells and the other directed at injured cells) to produce magnetic bifunctional cell engager (MagBICE). The antibodies link the therapeutic cells to the injured cells, whereas the iron core of MagBICE enables physical enrichment and imaging. We treat acute myocardial infarction by targeting exogenous bone marrow-derived stem cells (expressing CD45) or endogenous CD34-positive cells to injured cardiomyocytes (expressing myosin light chain. Targeting can be further enhanced by magnetic attraction, leading to augmented functional benefits. MagBICE represents a generalizable platform technology for regenerative medicine., Cell therapy requires sufficient amounts of therapeutic cells to be delivered to the injured tissue. Here the authors use magnetic iron nanoparticles conjugated with antibodies that bind therapeutic cells and cardiomyocytes to treat myocardial ischemia/reperfusion injury in rats and show that targeting to the heart is enhanced upon local application of a magnetic field.

Background: Self-assembling three-dimensional cardiospheres (CSps) are known to be effective in improving function and attenuating remodeling as adjunctive therapy in acute myocardial infarction (MI), and they exert functional benefits in large animals with convalescent MI. The effect of cardiospheres in chronically-remodeled myocardium post-MI, and the mechanisms of benefit are unknown. Methods: We performed permanent LAD ligation in Wistar Kyoto female rats. One month later the animals underwent repeat thoracotomy and were randomly assigned to one of two groups: vehicle-treated controls, or CSp-treated, where CSps (at a seeding dose of 2X10 6 cells) were injected in the peri-infarct area. Mechanisms were also probed in vitro . Results: CSps downregulated the canonical Tgfβ1-smad2/3 pathway in co-cultures with fibroblasts and reduced fibroblast proliferation in vitro . Soluble endoglin, abundantly expressed in CSps, was found to limit the Tgfβ1-induced fibrosis in a dose-dependent manner. In vivo , left ventricular function gradually increased one month post-CSp injection with further improvement after up to six months of follow up. Immunoblots verified Tgfβ1/smad cascade downregulation one month post-CSp injection and upregulation of soluble endoglin. In addition, scar mass was reduced one month post-CSp injection compared to controls. Collagen density was attenuated within the infarcted region of the treated group, while immunostaining showed enhanced vessel density in the same region. Six months post-transplantation, cardiomyocyte hypertrophy was reduced in the CSp-treated group compared to control. Conclusions: CSps injected into the peri-infarct zone enhance angiogenesis and reduce established fibrosis in chronically-infarcted myocardium, thereby reversing, at least partially, adverse remodeling of the heart. The underlying mechanism involves modification of Tgfβ1/smad signaling, a key pathway in the cardiac response to injury.

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit(+) cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit(+) cells amply generated cardiac endothelial cells. Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

Author(s): Yee, Kristine; Malliaras, Konstantinos; Kanazawa, Hideaki; Tseliou, Eleni; Cheng, Ke; Luthringer, Daniel J; Ho, Chak-Sum; Takayama, Kentaro; Minamino, Naoto; Dawkins, James F; Chowdhury, Supurna; Duong, Doan Trang; Seinfeld, Jeffrey; Middleton, Ryan C; Dharmakumar, Rohan; Li, Debiao; Marban, Linda; Makkar, Raj R; Marban, Eduardo | Abstract: BackgroundEpicardial injection of heart-derived cell products is safe and effective post-myocardial infarction (MI), but clinically-translatable transendocardial injection has never been evaluated. We sought to assess the feasibility, safety and efficacy of percutaneous transendocardial injection of heart-derived cells in porcine chronic ischemic cardiomyopathy.Methods and resultsWe studied a total of 89 minipigs; 63 completed the specified protocols. After NOGA-guided transendocardial injection, we quantified engraftment of escalating doses of allogeneic cardiospheres or cardiosphere-derived cells in minipigs (n = 22) post-MI. Next, a dose-ranging, blinded, randomized, placebo-controlled ("dose optimization") study of transendocardial injection of the better-engrafting product was performed in infarcted minipigs (n = 16). Finally, the superior product and dose (150 million cardiospheres) were tested in a blinded, randomized, placebo-controlled ("pivotal") study (n = 22). Contrast-enhanced cardiac MRI revealed that all cardiosphere doses preserved systolic function and attenuated remodeling. The maximum feasible dose (150 million cells) was most effective in reducing scar size, increasing viable myocardium and improving ejection fraction. In the pivotal study, eight weeks post-injection, histopathology demonstrated no excess inflammation, and no myocyte hypertrophy, in treated minipigs versus controls. No alloreactive donor-specific antibodies developed over time. MRI showed reduced scar size, increased viable mass, and attenuation of cardiac dilatation with no effect on ejection fraction in the treated group compared to placebo.ConclusionsDose-optimized injection of allogeneic cardiospheres is safe, decreases scar size, increases viable myocardium, and attenuates cardiac dilatation in porcine chronic ischemic cardiomyopathy. The decreases in scar size, mirrored by increases in viable myocardium, are consistent with therapeutic regeneration.

Aims Naturally secreted nanovesicles known as exosomes are required for the regenerative effects of cardiosphere-derived cells (CDCs), and exosomes mimic the benefits of CDCs in rodents. Nevertheless, exosomes have not been studied in a translationally realistic large-animal model. We sought to optimize delivery and assess the efficacy of CDC-secreted exosomes in pig models of acute (AMI) and convalescent myocardial infarction (CMI). Methods and results In AMI, pigs received human CDC exosomes (or vehicle) by intracoronary (IC) or open-chest intramyocardial (IM) delivery 30 min after reperfusion. No-reflow area and infarct size (IS) were assessed histologically at 48 h. Intracoronary exosomes were ineffective, but IM exosomes decreased IS from 80 ± 5% to 61 ± 12% ( P = 0.001) and preserved left ventricular ejection fraction (LVEF). In a randomized placebo-controlled study of CMI, pigs 4 weeks post-myocardial infarction (MI) underwent percutaneous IM delivery of vehicle ( n = 6) or CDC exosomes ( n = 6). Magnetic resonance imaging (MRI) performed before and 1 month after treatment revealed that exosomes (but not vehicle) preserved LV volumes and LVEF (−0.1 ± 2.2% vs. −5.4 ± 3.6%, P = 0.01) while decreasing scar size. Histologically, exosomes decreased LV collagen content and cardiomyocyte hypertrophy while increasing vessel density. Conclusion Cardiosphere-derived cell exosomes delivered IM decrease scarring, halt adverse remodelling and improve LVEF in porcine AMI and CMI. While conceptually attractive as cell-free therapeutic agents for myocardial infarction, exosomes have the disadvantage that IM delivery is necessary. [10.1093/eurheartj/ehw324][1] [1]: /lookup/doi/10.1093/eurheartj/ehw324

Ischemia and subsequent reperfusion (IR) produces injury to brain, eye and other tissues, contributing to the progression of important clinical pathologies. The response of cells to IR involves activation of several signaling pathways including those activating hypoxia and heat shock responsive transcription factors. However, specific roles of these responses in limiting cell damage and preventing cell death after IR have not been fully elucidated. Here, we have examined the role of heat shock factor 1 (HSF1) in the response of zebrafish embryos to hypoxia and subsequent return to normoxic conditions (HR) as a model for IR. Heat shock preconditioning elevated heat shock protein expression and protected zebrafish embryo eye and brain tissues against HR-induced apoptosis. These effects were inhibited by translational suppression of HSF1 expression. Reduced expression of HSF1 also increased cell death in brain and eye tissues of embryos subjected to hypoxia and reperfusion without prior heat shock. Surprisingly, reduced expression of HSF1 had only a modest effect on hypoxia-induced expression of Hsp70 and no effect on hypoxia-induced expression of Hsp27. These results establish the zebrafish embryo as a model for the study of ischemic injury in the brain and eye and reveal a critical role for HSF1 in the response of these tissues to HR. Our results also uncouple the role of HSF1 expression from that of Hsp27, a well characterized heat shock protein considered essential for cell survival after hypoxia. Alternative roles for HSF1 are considered.

Kohonen self-organizing neural networks, also called self-organizing maps (SOMs), have been used successfully to recognize human phonemes and in this way to aid in human speech recognition. This paper describes how SOMS also can be used to associate specific information content with animal vocalizations. A SOM was used to identify acoustic units in Gunnison’s prairie dog alarm calls that were vocalized in the presence of three different predator species. Some of these acoustic units and their combinations were found exclusively in the alarm calls associated with a particular predator species and were used to associate predator species information with individual alarm calls. This methodology allowed individual alarm calls to be classified by predator species with an average of 91% accuracy. Furthermore, the topological structure of the SOM used in these experiments provided additional insights about the acoustic units and their combinations that were used to classify the target alarm calls. An important benefit of the methodology developed in this paper is that it could be used to search for groups of sounds associated with information content for any animal whose vocalizations are composed of multiple simultaneous frequency components.