Michelle Kreke

Exosomes secreted by cardiosphere-derived cells reduce scarring, attenuate adverse remodelling, and improve function in acute and chronic porcine myocardial infarction.

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.

Validation of contrast-enhanced magnetic resonance imaging to monitor regenerative efficacy after cell therapy in a porcine model of convalescent myocardial infarction

Background— Magnetic resonance imaging (MRI) in the CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trial revealed that cardiosphere-derived cells (CDCs) decrease scar size and increase viable myocardium after myocardial infarction (MI), but MRI has not been validated as an index of regeneration after cell therapy. We tested the validity of contrast-enhanced MRI in quantifying scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. Methods and Results— Yucatan minipigs underwent induction of MI and 2–3 weeks later were randomized to receive intracoronary infusion of 12.5×106 mismatched allogeneic CDCs or vehicle. Allogeneic CDCs induced mild local mononuclear infiltration but no systemic immunogenicity. MRI revealed that allogeneic CDCs attenuated remodeling, improved global and regional function, decreased scar size, and increased viable myocardium compared with placebo 2 months post-treatment. Extensive histological analysis validated quantitatively the MRI measurements of scar size, scar mass, and viable mass. CDCs neither altered gadolinium contrast myocardial kinetics nor induced changes in vascular density or architecture in viable and scarred myocardium. Histology demonstrated that CDCs lead to cardiomyocyte hyperplasia in the border zone, consistent with the observed stimulation of endogenous regenerative mechanisms (cardiomyocyte cycling, upregulation of endogenous progenitors, angiogenesis). Conclusions— Contrast-enhanced MRI accurately measures scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. MRI represents a useful tool for assessing dynamic changes in the infarct and monitoring regenerative efficacy.

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

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.

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

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).

Cardiospheres and cardiosphere-derived cells as therapeutic agents following myocardial infarction.

Heart disease is a major cause of morbidity and mortality. Cellular therapies hold significant promise for patients with heart disease. Heart-derived progenitor cells are capable of repairing a diseased heart through modulation of growth factor milieu and temporary engraftment leading to endogenous repair. The proof-of-concept CADUCEUS clinical trial using cardiosphere-derived cells has shown evidence of therapeutic cardiac tissue regeneration. Future clinical trials are now being planned to generate additional safety and efficacy data in the hopes of building toward an approved cellular therapy for heart disease.

Intracoronary Delivery of Self-Assembling Heart-Derived Microtissues (Cardiospheres) for Prevention of Adverse Remodeling in a Pig Model of Convalescent Myocardial Infarction

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 &mgr;m), 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 (1.3×106) 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.

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

Background 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. Methods and Results 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. 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.

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.

Validation of Contrast-Enhanced MRI to Monitor Regenerative Efficacy after Cell Therapy in a Porcine Model of Convalescent Myocardial Infarction

Background— Magnetic resonance imaging (MRI) in the CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trial revealed that cardiosphere-derived cells (CDCs) decrease scar size and increase viable myocardium after myocardial infarction (MI), but MRI has not been validated as an index of regeneration after cell therapy. We tested the validity of contrast-enhanced MRI in quantifying scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. Methods and Results— Yucatan minipigs underwent induction of MI and 2–3 weeks later were randomized to receive intracoronary infusion of 12.5×106 mismatched allogeneic CDCs or vehicle. Allogeneic CDCs induced mild local mononuclear infiltration but no systemic immunogenicity. MRI revealed that allogeneic CDCs attenuated remodeling, improved global and regional function, decreased scar size, and increased viable myocardium compared with placebo 2 months post-treatment. Extensive histological analysis validated quantitatively the MRI measurements of scar size, scar mass, and viable mass. CDCs neither altered gadolinium contrast myocardial kinetics nor induced changes in vascular density or architecture in viable and scarred myocardium. Histology demonstrated that CDCs lead to cardiomyocyte hyperplasia in the border zone, consistent with the observed stimulation of endogenous regenerative mechanisms (cardiomyocyte cycling, upregulation of endogenous progenitors, angiogenesis). Conclusions— Contrast-enhanced MRI accurately measures scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. MRI represents a useful tool for assessing dynamic changes in the infarct and monitoring regenerative efficacy.

Erratum: Cardiospheres and cardiosphere-derived cells as therapeutic agents following myocardial infarction (Expert Review of Cardiovascular Therapy (2012) 10:9 (1185-1194))

Following the publication of the Review by Michelle Kreke, Rachel Ruckdeschel Smith, Linda Marbán and Eduardo Marbán, ‘Cardiospheres and cardiosphere-derived cells as therapeutic agents following myocardial infarction’, published in the September 2012 issue of Expert Review of Cardiovascular Therapy (Expert Rev. Cardiovasc. Ther. 10[9], 1185–1194 [2012]), it has been brought to our attention that the websites on page 1194 were incorrectly printed as: