Cellular Postconditioning: Allogeneic Cardiosphere-Derived Cells Reduce Infarct Size and Attenuate Microvascular Obstruction When Administered After Reperfusion in Pigs With Acute Myocardial Infarction

Intracoronary (IC) delivery of cardiosphere-derived cells (CDCs) has been demonstrated to be safe and effective in porcine and human chronic myocardial infarction (MI). However, IC delivery of CDCs after reperfusion in acute MI has never been assessed in a clinically-relevant large animal model. We tested CDCs as adjunctive therapy to reperfusion in a porcine model of MI

Y RNA fragment in extracellular vesicles confers cardioprotection via modulation of IL‐10 expression and secretion

Abstract Cardiosphere‐derived cells (CDCs) reduce myocardial infarct size via secreted extracellular vesicles (CDC‐EVs), including exosomes, which alter macrophage polarization. We questioned whether short non‐coding RNA species of unknown function within CDC‐EVs contribute to cardioprotection. The most abundant RNA species in CDC‐EVs is a Y RNA fragment (EV‐YF1); its relative abundance in CDC‐EVs correlates with CDC potency in vivo. Fluorescently labeled EV‐YF1 is actively transferred from CDCs to target macrophages via CDC‐EVs. Direct transfection of macrophages with EV‐YF1 induced transcription and secretion of IL‐10. When cocultured with rat cardiomyocytes, EV‐YF1‐primed macrophages were potently cytoprotective toward oxidatively stressed cardiomyocytes through induction of IL‐10. In vivo, intracoronary injection of EV‐YF1 following ischemia/reperfusion reduced infarct size. A fragment of Y RNA, highly enriched in CDC‐EVs, alters Il10 gene expression and enhances IL‐10 protein secretion. The demonstration that EV‐YF1 confers cardioprotection highlights the potential importance of diverse exosomal contents of unknown function, above and beyond the usual suspects (e.g., microRNAs and proteins)

Widespread Myocardial Delivery of Heart-Derived Stem Cells by Nonocclusive Triple-Vessel Intracoronary Infusion in Porcine Ischemic Cardiomyopathy: Superior Attenuation of Adverse Remodeling Documented by Magnetic Resonance Imaging and Histology.

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

Structure-function correlations.

<p><b>A</b>. Representative 2,3,5-triphenyltetrazoliumchloride (TTC) sections covering the LV chamber from the apex to the base from all three groups tested. The blue arrowhead indicates the section level that corresponds to the MR images in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144523#pone.0144523.g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144523#pone.0144523.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144523#pone.0144523.g005" target="_blank">5</a>. <b>B</b>. Correlation between the scar mass (g) evaluated separately by LGE images (JD) and by TTC staining (ET). <b>C</b>. Representative tagged CMR images with infarct zone (IZ) defined by the presence of enhancing myocardium, neighboring myocardium as border zone (BZ), and normal myocardium as remote zone (RZ) for the analysis of regional strain, highlighting the contractility differences among the groups. Blue arrowheads indicate the RV insertion and the location of the first segment of each map. <b>D., E., F</b>. Representative diagrams of the averaged strain included in the analysis (the more negative, the greater the contractility). <b>G</b>. Better contractility was observed in the CDC-treated group compared to the placebo. <b>H</b>. Trend towards better effect of CDC therapy on mid-ventricular wall synchrony assessed by CURE ratio. Error bars indicate SEM. * p<0.05, ** p<0.01</p

Multi-vessel study design and safety.

<p><b>A</b>. Timeline of the study. <b>B</b>., Allogeneic CDC isolation and manufacturing from 2 male donor hearts resulted in a master cell bank and bags of frozen CDCs. <b>C</b>. Effects of cryopreservation on CD105 and CD45 expression of CDCs. <b>D., E., F</b>. TIMI flow post-infusion in each vessel. <b>G., H</b>. TnI bump (7.4ng/ml) was observed at 24hrs in one animal after the stop-flow protocol. Error bars indicate SEM. Abbreviations: MI, myocardial infarction; CDCs, cardiosphere-derived cells; MRI, magnetic resonance imaging; LAD: left anterior descending artery.</p

Single-vessel Study.

<p><b>A</b>. Illustration of stop-flow protocol and <b>B</b>., the continuous-flow protocol. <b>C, D</b>. Box Plots showing serum [TnI] before infusion and 24hrs post infusion (p = ns; normal <0.05ng/ml). <b>E</b>. Short-axis contrast-enhanced images at baseline and 4 weeks after CDC or vehicle infusion showing <b>F</b>. significant changes of ejection fraction in placebo versus either treated group (p = 0.03 placebo vs CDCs stop-flow and p<0.01 placebo vs CDCs continuous-flow). <b>G</b>. Changes in end-systolic volume between baseline and 4 weeks post-infusion (p = 0.02 placebo vs CDCs stop-flow and p = 0.05 placebo vs CDCs continuous-flow), and <b>H</b>., in end-diastolic volume (p = 0.06 placebo vs CDCs stop-flow and p = 0.2 placebo vs CDCs continuous-flow). Changes in scar mass (<b>I</b>, p = 0.08 placebo vs CDCs stop-flow and p<0.01 placebo vs CDCs continuous-flow), <b>J</b>., infarct wall motion (p = 0.04 placebo vs CDCs stop-flow and p = 0.02 placebo vs CDCs continuous-flow) and <b>K</b>., infarct wall thickness between placebo and treated groups (p = ns). Error bars indicate SEM. * p<0.05, ** p<0.01</p

Durable Benefits of Cellular Postconditioning: Long‐Term Effects of Allogeneic Cardiosphere‐Derived Cells Infused After Reperfusion in Pigs with Acute Myocardial Infarction

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

Side-by-side evaluation of intramyocardial injections and intracoronary infusions of CDCs data.

<p><b>A</b>. The cell treatment effect of both intramyocardial and intracoronary delivery was superior to placebo with no difference between the 2 treatment groups. <b>B</b>. Similar changes were observed in scar size reduction. <b>C</b>. More cells were detected post intramyocardial injections compared to intracoronary delivery at 24hr of evaluation. Intracoronary data from the present study; intramyocardial data replotted from ref. 32. Error bars indicate SEM.</p

Vessel density and cardiomyocyte hypertrophy.

<p><b>A</b>. Representative immunostaining sections with sma, isolectin B4 and DAPI, 1 month post treatment. <b>B</b>. Vessel density within the IZ (p = 0.05), <b>C</b>., the BZ and <b>D</b>., the RZ in the CDC-treated compared to placebo (Scale bar = 75μm). <b>E</b>. Representative sections immunostained with α-sa and WGA. <b>F., G</b>. Similar cardiomyocyte diameters in all three groups, both in the BZ and in the RZ. Scale bar = 50μm. Abbreviations: IZ, infarct zone; BZ, border zone; RZ, remote zone; sma, smooth muscle actin (red); isolectin B4 (green); DAPI, blue, 4',6-diamidino-2-phenylindole; asa, α-sarcomeric actinin; WGA, wheat germ agglutinin to define the cell borders. Error bars indicate SEM.</p