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BACKGROUND: Enteric neuropathies, which result from abnormalities of the enteric nervous system, are associated with significant morbidity and high health-care costs, but current treatments are unsatisfactory. Cell-based therapy offers an innovative approach to replace the absent or abnormal enteric neurons and thereby restore gut function. METHODS: Enteric neuronal stem cells (ENSCs) were isolated from the gastrointestinal tract of Wnt1-Cre;R26tdTomato mice and generated neurospheres (NS). NS transplants were performed via injection into the mid-colon mesenchyme of nNOS-/- mouse, a model of colonic dysmotility, using either 1 (n = 12) or 3 (n = 12) injections (30 NS per injection) targeted longitudinally 1-2 mm apart. Functional outcomes were assessed up to 6 weeks later using electromyography (EMG), electrical field stimulation (EFS), optogenetics, and by measuring colorectal motility. RESULTS: Transplanted ENSCs formed nitrergic neurons in the nNOS-/- recipient colon. Multiple injections of ENSCs resulted in a significantly larger area of coverage compared to single injection alone and were associated with a marked improvement in colonic function, demonstrated by (1) increased colonic muscle activity by EMG recording, (2) faster rectal bead expulsion, and (3) increased fecal pellet output in vivo. Organ bath studies revealed direct neuromuscular communication by optogenetic stimulation of channelrhodopsin-expressing ENSCs and restoration of smooth muscle relaxation in response to EFS. CONCLUSIONS: These results demonstrate that transplanted ENSCs can form effective neuromuscular connections and improve colonic motor function in a model of colonic dysmotility, and additionally reveal that multiple sites of cell delivery led to an improved response, paving the way for optimized clinical trial design.

The enteric nervous system (ENS) is an extensive network of neurons and glia within the wall of the gastrointestinal (GI) tract that regulates many essential GI functions. Consequently, disorders of the ENS due to developmental defects, inflammation, infection, or age-associated neurodegeneration lead to serious neurointestinal diseases. Despite the prevalence and severity of these diseases, effective treatments are lacking as they fail to directly address the underlying pathology. Neuronal stem cell therapy represents a promising approach to treating diseases of the ENS by replacing the absent or injured neurons, and an autologous source of stem cells would be optimal by obviating the need for immunosuppression. We utilized the swine model to address key questions concerning cell isolation, delivery, engraftment, and fate in a large animal relevant to human therapy. We successfully isolated neural stem cells from a segment of small intestine resected from 1-month-old swine. Enteric neuronal stem cells (ENSCs) were expanded as neurospheres that grew optimally in low-oxygen (5%) culture conditions. Enteric neuronal stem cells were labeled by lentiviral green fluorescent protein (GFP) transduction, then transplanted into the same swine from which they had been harvested. Endoscopic ultrasound was then utilized to deliver the ENSCs (10,000-30,000 neurospheres per animal) into the rectal wall. At 10 and 28 days following injection, autologously derived ENSCs were found to have engrafted within rectal wall, with neuroglial differentiation and no evidence of ectopic spreading. These findings strongly support the feasibility of autologous cell isolation and delivery using a clinically useful and minimally invasive technique, bringing us closer to first-in-human ENSC therapy for neurointestinal diseases.

BackgroundAlthough ∼20% of the elderly population develops atrial fibrillation (AF), little is known about the mechanisms. Heart failure with preserved ejection fraction (HFpEF), which is associated with AF, is more common in aged women than in men.ObjectiveThe purpose of this study was to identify potential mechanisms of AF in an age-related HFpEF model.MethodsIn aged female Fischer F344 rats (21- to 24-month-old), which are prone to HFpEF, we induced AF by atrial pacing. Young Fischer F344 female rats (3- to 4-month-old) and age-matched Sprague Dawley female rats (27-month-old) served as controls. Phenotyping included echocardiography to assess left ventricular structure/function; in vivo electrophysiology and ex vivo high-resolution optical mapping to assess AF vulnerability; systemic and atrial inflammatory profiling; atrial histology; and expression of inflammasome signaling proteins.ResultsAged rats developed left ventricular hypertrophy, left atrial enlargement, diastolic dysfunction, and pulmonary congestion, without ejection fraction impairment, thus meeting the criteria for HFpEF. Increased serum inflammatory markers, hypertension, and obesity further characterize aged females. Sinoatrial and atrioventricular node dysfunction was associated with the high inducibility of AF in aged rats. Ex vivo electrical activation mapping revealed abnormal β-adrenergic responsiveness and slowed conduction velocity. Atrial inflammasome signaling was enhanced in aged rats, which may contribute to fibrotic remodeling and high AF susceptibility.ConclusionTogether, our data demonstrate that aging-related atrial remodeling and HFpEF are associated with atrial enlargement, fibrosis, conduction abnormalities, and nodal dysfunction, favoring a substrate conducive to AF.

Sudden death is the most common mode of exodus in patients with heart failure and preserved ejection fraction (HFpEF). Cardiosphere-derived cells (CDCs) reduce inflammation and fibrosis in a rat model of HFpEF, improving diastolic function and prolonging survival. We tested the hypothesis that CDCs decrease ventricular arrhythmias (VAs) and thereby possibly contribute to prolonged survival. Dahl salt-sensitive rats were fed a high-salt diet to induce HFpEF. Allogeneic rat CDCs (or phosphate-buffered saline as placebo) were injected in rats with echo-verified HFpEF. CDC-injected HFpEF rats were less prone to VA induction by programmed electrical stimulation. Action potential duration (APD) was shortened, and APD homogeneity was increased by CDC injection. Transient outward potassium current density was upregulated in cardiomyocytes from CDC rats relative to placebo, as were the underlying transcript (Kcnd3) and protein (Kv4.3) levels. Fibrosis was attenuated in CDC-treated hearts, and survival was increased. Sudden death risk also trended down, albeit nonsignificantly. CDC therapy decreased VA in HFpEF rats by shortening APD, improving APD homogeneity, and decreasing fibrosis. Unlike other stem/progenitor cells, which often exacerbate arrhythmias, CDCs reverse electrical remodeling and suppress arrhythmogenesis in HFpEF.

BackgroundHeart failure with preserved ejection fraction (HFpEF) represents approximately half of heart failure, and its incidence continues to increase. The leading cause of mortality in HFpEF is sudden death, but little is known about the underlying mechanisms.MethodsDahl salt-sensitive rats were fed a high-salt diet (8% NaCl) from 7 weeks of age to induce HFpEF (n=38). Rats fed a normal-salt diet (0.3% NaCl) served as controls (n=13). Echocardiograms were performed to assess systolic and diastolic function from 14 weeks of age. HFpEF-verified and control rats underwent programmed electrical stimulation. Corrected QT interval was measured by surface ECG. The mechanisms of ventricular arrhythmias (VA) were probed by optical mapping, whole-cell patch clamp to measure action potential duration and ionic currents, and quantitative polymerase chain reaction and Western blotting to investigate changes in ion channel expression.ResultsAfter 7 weeks of a high-salt diet, 31 of 38 rats showed diastolic dysfunction and preserved ejection fraction along with signs of heart failure and hence were diagnosed with HFpEF. Programmed electric stimulation demonstrated increased susceptibility to VA in HFpEF rats (P

Infusion of allogeneic cardiosphere-derived cells (allo-CDCs) postreperfusion elicits cardioprotective cellular postconditioning in pigs with acute myocardial infarction. However, the long-term effects of allo-CDCs have not been assessed. We performed a placebo-controlled pivotal study for long-term evaluation, as well as shorter-term mechanistic studies. Minipigs underwent 1.5-hour mid-left anterior descending balloon occlusion followed by reperfusion and were randomized to receive intracoronary allo-CDCs 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-CDCs 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-CDCs did not evoke lymphohistiocytic infiltration or systemic humoral memory response. Short-term experiments designed to probe mechanism revealed antiapoptotic effects of allo-CDCs on cardiomyocytes and increases in cytoprotective macrophages, but no increase in overall inflammatory cell infiltration 2 hours after cell therapy. 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.

Ischemic injury in the heart induces an inflammatory cascade that both repairs damage and exacerbates scar tissue formation. Cardiosphere-derived cells (CDCs) are a stem-like population that is derived ex vivo from cardiac biopsies; they confer both cardioprotection and regeneration in acute myocardial infarction (MI). While the regenerative effects of CDCs in chronic settings have been studied extensively, little is known about how CDCs confer the cardioprotective process known as cellular postconditioning. Here, we used an in vivo rat model of ischemia/reperfusion (IR) injury-induced MI and in vitro coculture assays to investigate how CDCs protect stressed cardiomyocytes. Compared with control animals, animals that received CDCs 20 minutes after IR had reduced infarct size when measured at 48 hours. CDCs modified the myocardial leukocyte population after ischemic injury. Specifically, introduction of CDCs reduced the number of CD68+ macrophages, and these CDCs secreted factors that polarized macrophages toward a distinctive cardioprotective phenotype that was not M1 or M2. Systemic depletion of macrophages with clodronate abolished CDC-mediated cardioprotection. Using both in vitro coculture assays and a rat model of adoptive transfer after IR, we determined that CDC-conditioned macrophages attenuated cardiomyocyte apoptosis and reduced infarct size, thereby recapitulating the beneficial effects of CDC therapy. Together, our data indicate that CDCs limit acute injury by polarizing an effector macrophage population within the heart.

BackgroundIntracoronary 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 resultsFirst, 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.ConclusionsIntracoronary 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.

Chromatin remodeling, particularly histone acetylation, plays a critical role in the progression of pathological cardiac hypertrophy and heart failure. We hypothesized that curcumin, a natural polyphenolic compound abundant in the spice turmeric and a known suppressor of histone acetylation, would suppress cardiac hypertrophy through the disruption of p300 histone acetyltransferase-dependent (p300-HAT--dependent) transcriptional activation. We tested this hypothesis using primary cultured rat cardiac myocytes and fibroblasts as well as two well-established mouse models of cardiac hypertrophy. Curcumin blocked phenylephrin-induced (PE-induced) cardiac hypertrophy in vitro in a dose-dependent manner. Furthermore, curcumin both prevented and reversed mouse cardiac hypertrophy induced by aortic banding (AB) and PE infusion, as assessed by heart weight/BW and lung weight/BW ratios, echocardiographic parameters, and gene expression of hypertrophic markers. Further investigation demonstrated that curcumin abrogated histone acetylation, GATA4 acetylation, and DNA-binding activity through blocking p300-HAT activity. Curcumin also blocked AB-induced inflammation and fibrosis through disrupting p300-HAT--dependent signaling pathways. Our results indicate that curcumin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through suppression of p300-HAT activity and downstream GATA4, NF-[kappa]B, and TGF-[beta]-Smad signaling pathways., Introduction Cardiac hypertrophy is an adaptive enlargement of the myocardium in response to increased workload, characterized by an increase in the size of individual cardiac myocytes and whole-organ enlargement. Although [...]

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-reflowarea 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 (20.1±2.2% vs. 25.4±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. [ABSTRACT FROM AUTHOR]