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

Allogeneic Cardiospheres Delivered via Percutaneous Transendocardial Injection Increase Viable Myocardium, Decrease Scar Size, and Attenuate Cardiac Dilatation in Porcine Ischemic Cardiomyopathy

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

Identification of a Sudden Cardiac Death Susceptibility Locus at 2q24.2 through Genome-Wide Association in European Ancestry Individuals

Sudden cardiac death (SCD) continues to be one of the leading causes of mortality worldwide, with an annual incidence estimated at 250,000–300,000 in the United States and with the vast majority occurring in the setting of coronary disease. We performed a genome-wide association meta-analysis in 1,283 SCD cases and >20,000 control individuals of European ancestry from 5 studies, with follow-up genotyping in up to 3,119 SCD cases and 11,146 controls from 11 European ancestry studies, and identify the BAZ2B locus as associated with SCD (P = 1.8×10−10). The risk allele, while ancestral, has a frequency of ∼1.4%, suggesting strong negative selection and increases risk for SCD by 1.92–fold per allele (95% CI 1.57–2.34). We also tested the role of 49 SNPs previously implicated in modulating electrocardiographic traits (QRS, QT, and RR intervals). Consistent with epidemiological studies showing increased risk of SCD with prolonged QRS/QT intervals, the interval-prolonging alleles are in aggregate associated with increased risk for SCD (P = 0.006)

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)

Angiogenesis, Cardiomyocyte Proliferation and Anti-Fibrotic Effects Underlie Structural Preservation Post-Infarction by Intramyocardially-Injected Cardiospheres

<div><p>Objective</p><p>We sought to understand the cellular and tissue-level changes underlying the attenuation of adverse remodeling by cardiosphere transplantation in acute myocardial infarction (MI).</p><p>Background</p><p>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.</p><p>Methods</p><p>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).</p><p>Results</p><p><i>In vivo</i> 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. <i>In vitro</i>, CSp-conditioned media reduced collagen production in coculture with fibroblasts and triggered neoangiogenesis in an <i>ex vivo</i> aortic ring assay.</p><p>Conclusion</p><p>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.</p></div

Metalloproteinase activity.

<p>(A) Immunoblotting (top 10) and zymograms (bottom membrane) were performed to examine the relative abundance of representative MMP types in myocardial tissue obtained from the peri-infarct zone 7 days post MI and CSp injection. For MMP2 and MMP13 the same membrane was used. For MMP9, MMP3 and TIMP2 the same membrane was used as well. The same samples were used in both membranes. All three samples in each group were evaluated. (B,E,F) Increases in MMP2, MMP13, and TIMP2 were observed in the peri-infarct zone of the post CSp transplanted myocardium. (C) In contrast, relative levels of MMP9 increased in the control compared to the treated group. (D,G) No differences were detected for MT1-MMP and MMP3. Data are mean±SD. ¶ p<0.05 control vs. MI+CSp, * p<0.05 vs. sham.</p

Aortic ring sprouts.

<p>Representative phase contrast images from aortic rings embedded in collagen matrices 9 days post treatment with (A) CSp conditioned media, (B) 10% FBS culture media and (C) endothelial basal media. (D) Quantification of sprouts on day 9 under phase contrast microscopy. Arrows point at the new sprouts. (E) Immunofluorescence of the CSp conditioned media treated aortic ring reveals the phenotype of the de novo formed microvessels. Inserted photo is a high power field image of the immunostained microvessels for BS-lectin and smooth muscle actin. Three different experiments per group were evaluated (Scale bars 10 µm). Data are mean±SD. ¶ p<0.05 control vs. MI+CSp.</p

Cardiac tissue structure.

<p>(A) Representative photomicrographs of immunohistochemical staining of smooth muscle actin, wheat germ agglutinin, and Dapi in myocardial tissue sections. Five different heart tissues per group were stained. Approximately 10–15 high-power fields per area from a minimum 3 slides per heart were analyzed to obtain the average regional cross sectional area and regional myocyte nuclear density. 100 cardiomyocytes per heart were evaluated. (B) Quantification of cross-sectional area in the peri-infarct and D. in the remote zone respectively. (C,E) Measurement of the total number of cell nuclei per field evaluated in both above-mentioned regions. (F) Representative photomicrographs of immunohistochemical staining of α-sarcomeric actin, Ki67 and Dapi, in the peri-infarct zone and (I) in the remote area. (G–H) Quantification of proliferating cardiomyocytes per total number of cardiomyocytes per field and of proliferating cardiomyocytes per total nuclei per field respectively. (J–K) Same quantification as in G–H but in the remote area. Arrows point to Ki67+/asa+ cells. (Scale bars 50 µm). Data are mean±SEM. ¶ p<0.05 control vs. MI+CSp, * p<0.05 vs. sham.</p

Tissue morphology and cardiac function.

<p>(A) Representative photomicrographs of myocardial sections stained with Masson's trichrome. Sections are from CSp treated (n = 5) and placebo treated (n = 5) animals at 7 and 21 days post MI and treatment. (B–F) Quantitative data for infarct mass, viable mass, infarct size, infarct thickness and septum thickness. Five different hearts were processed and 5–6 different sections from the apex to the base from each heart were used for the analysis. (G–I) EF ejection fraction, LVEDD left ventricular end diastolic diameter, LVPWT left ventricular posterior wall thickness in diastole. Baseline represents measurements 18–24 hours after coronary ligation and treatment. n = 6 for each animal group studied. Bars are presented as mean±SD. ¶ p<0.05 control vs MI +CSp.</p