Eleni Tseliou

Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart

Cardiosphere‐derived cells (CDCs) have been shown to regenerate infarcted myocardium in patients after myocardial infarction (MI). However, whether the cells of the newly formed myocardium originate from the proliferation of adult cardiomyocytes or from the differentiation of endogenous stem cells remains unknown. Using genetic fate mapping to mark resident myocytes in combination with long‐term BrdU pulsing, we investigated the origins of postnatal cardiomyogenesis in the normal, infarcted and cell‐treated adult mammalian heart. In the normal mouse heart, cardiomyocyte turnover occurs predominantly through proliferation of resident cardiomyocytes at a rate of ∼1.3–4%/year. After MI, new cardiomyocytes arise from both progenitors as well as pre‐existing cardiomyocytes. Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium. The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair. Thus, CDCs induce myocardial regeneration by differentially upregulating two mechanisms of endogenous cell proliferation.

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.

Macrophages mediate cardioprotective cellular postconditioning in acute myocardial infarction

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.

Allogeneic Cardiospheres Safely Boost Cardiac Function and Attenuate Adverse Remodeling After Myocardial Infarction in Immunologically Mismatched Rat Strains

OBJECTIVES We sought to characterize the immunologic profile of allogeneic cardiospheres, which are 3-dimensional, self-assembling, cardiac-derived microtissues, and to evaluate their safety and efficacy in repairing ischemic heart tissue. BACKGROUND Intramyocardial injection of autologous cardiospheres ameliorates remodeling and improves global function in infarcted myocardium. It is as yet unknown whether allogeneic cardiospheres are similarly effective without eliciting deleterious immune reactions. METHODS We expanded cardiospheres from male Wistar Kyoto rat hearts and injected them surgically in the peri-infarct zone of Wistar Kyoto (syngeneic group, n = 28) and Brown Norway female rats (allogeneic group, n = 29). Female rats from both strains (n = 37) injected with normal saline served as controls. RESULTS In vitro, cardiospheres expressed a low immunogenic profile and inhibited proliferation of alloreactive T cells. In vivo, cell engraftment was similar in the syngeneic and allogeneic groups 1 week and 3 weeks after transplantation. Reductions in scar size and scar collagen content and increases in viable mass in the risk region were accompanied by improvements in left ventricular function and attenuation of left ventricle remodeling that were sustained during 6 months of follow up. Transplantation of allogeneic cardiospheres increased tissue expression of the regenerative growth factors vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1, stimulating angiogenesis. Syngeneic and allogeneic cardiospheres attenuated the inflammatory response observed histologically in the peri-infarct region. CONCLUSIONS Allogeneic cardiospheres increase viable myocardium, decrease scar, improve function, and attenuate adverse remodeling in the infarcted rat heart, without deleterious immunological sequelae. These observations lay the groundwork for developing cardiospheres as a novel off-the-shelf microtissue product for myocardial regeneration.

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.

Stimulation of endogenous cardioblasts by exogenous cell therapy after myocardial infarction

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.

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.

Exhaled Nitric Oxide and Exhaled Breath Condensate pH in Severe Refractory Asthma

BACKGROUND Distinct inflammatory cellular phenotypes of severe refractory asthma (SRA) have been reported. Fractional exhaled nitric oxide (FeNO) primarily is related to eosinophilic inflammation. Exhaled breath condensate (EBC) pH has been suggested as a noninvasive tool in the assessment of patients with asthma. We sought to determine whether FeNO and EBC pH could identify the presence and type of the underlying cellular inflammation in patients with SRA. METHODS Twenty-nine patients with SRA, 27 patients with moderate asthma, and 17 healthy subjects underwent FeNO measurement, EBC collection for pH measurement, and sputum induction for cell count identification. RESULTS FeNO was significantly higher and pH significantly lower in patients with SRA than in the other groups. In SRA, FeNO levels of > 19 parts per billion were associated with a sensitivity of 0.78 and a specificity of 0.73 for sputum eosinophilia, whereas FeNO levels of < 19 parts per billion were associated with a sensitivity of 0.63 and a specificity of 0.9 for sputum neutrophilia irrespective of the presence of eosinophils. The pH failed to predict the cellular profile in SRA, but a cutoff value of < 7.37 could predict sputum eosinophilia in moderate asthma. CONCLUSIONS In patients with SRA, different FeNO threshold values can identify those with predominant eosinophilia as well as those with neutrophilia. FeNO levels were reduced in patients with predominant neutrophilia regardless of the concomitant presence of eosinophilia. Although pH could not identify the cellular profile in SRA, it seemed to be a better index for predicting eosinophilia in moderate asthma.

Therapeutic efficacy of cardiosphere-derived cells in a transgenic mouse model of non-ischaemic dilated cardiomyopathy

AIM Cardiosphere-derived cells (CDCs) produce regenerative effects in the post-infarct setting. However, it is unclear whether CDCs are beneficial in non-ischaemic dilated cardiomyopathy (DCM). We tested the effects of CDC transplantation in mice with cardiac-specific Gαq overexpression, which predictably develop progressive cardiac dilation and failure, with accelerated mortality. METHODS AND RESULTS Wild-type mouse CDCs (10(5) cells) or vehicle only were injected intramyocardially in 6-, 8-, and 11-week-old Gαq mice. Cardiac function deteriorated in vehicle-treated mice over 3 months of follow-up, accompanied by oxidative stress, inflammation and adverse ventricular remodelling. In contrast, CDCs preserved cardiac function and volumes, improved survival, and promoted cardiomyogenesis while blunting Gαq-induced oxidative stress and inflammation in the heart. The mechanism of benefit is indirect, as long-term engraftment of transplanted cells is vanishingly low. CONCLUSIONS Cardiosphere-derived cells reverse fundamental abnormalities in cell signalling, prevent adverse remodelling, and improve survival in a mouse model of DCM. The ability to impact favourably on disease progression in non-ischaemic heart failure heralds new potential therapeutic applications of CDCs.

Angiogenesis, cardiomyocyte proliferation and anti-fibrotic effects underlie structural preservation post-infarction by intramyocardially-injected cardiospheres.

Objective We sought to understand the cellular and tissue-level changes underlying the attenuation of adverse remodeling by cardiosphere transplantation in acute myocardial infarction (MI). Background Cardiospheres (CSps) are heart-derived multicellular clusters rich in stemness and capable of multilineage differentiation. Post-MI CSp transplantation improves left ventricular (LV) function and attenuates remodeling in both small and large animal studies. However, the mechanisms of benefit have not yet been fully elucidated. Methods Four groups were studied: 1) “Sham” (Wistar Kyoto rats with thoracotomy and ligature without infarction); 2) “MI” (proximal LAD ligation with peri-infarct injection of vehicle); 3) “MI+CSp” (MI with cardiospheres injected in the peri-infarct area); 4) “Small MI” (mid-LAD ligation only). Results In vivo 1 week after CSp transplantation, LV functional improvement was associated with an increase in cardiomyocyte proliferation. By 3 weeks, microvessel formation was enhanced, while cardiomyocyte hypertrophy and regional fibrosis were attenuated. Collagen deposition was reduced, collagen degradation was enhanced, and MMPs were upregulated. The beneficial effects of CSp transplantation were not observed in the Small MI group, indicating that the effects are not solely due to CSp-induced cardioprotection. In vitro, CSp-conditioned media reduced collagen production in coculture with fibroblasts and triggered neoangiogenesis in an ex vivo aortic ring assay. Conclusion Cardiospheres enhance cardiomyocyte proliferation and angiogenesis, and attenuate hypertrophy and fibrosis, in the ischemic myocardium. These synergistic effects underlie the attenuation of adverse remodeling by cardiospheres.