Cell-Based HIF1α Gene Therapy Reduces Myocardial Scar and Enhances Angiopoietic Proteome, Transcriptomic and miRNA Expression in Experimental Chronic Left Ventricular Dysfunction

Recent preclinical investigations and clinical trials with stem cells mostly studied bone-marrow-derived mononuclear cells (BM-MNCs), which so far failed to meet clinically significant functional study endpoints. BM-MNCs containing small proportions of stem cells provide little regenerative potential, while mesenchymal stem cells (MSCs) promise effective therapy via paracrine impact. Genetic engineering for rationally enhancing paracrine effects of implanted stem cells is an attractive option for further development of therapeutic cardiac repair strategies. Non-viral, efficient transfection methods promise improved clinical translation, longevity and a high level of gene delivery. Hypoxia-induced factor 1α is responsible for pro-angiogenic, anti-apoptotic and anti-remodeling mechanisms. Here we aimed to apply a cellular gene therapy model in chronic ischemic heart failure in pigs. A non-viral circular minicircle DNA vector (MiCi) was used for in vitro transfection of porcine MSCs (pMSC) with HIF1α (pMSC-MiCi-HIF-1α). pMSCs-MiCi-HIF-1α were injected endomyocardially into the border zone of an anterior myocardial infarction one month post-reperfused-infarct. Cell injection was guided via 3D-guided NOGA electro-magnetic catheter delivery system. pMSC-MiCi-HIF-1α delivery improved cardiac output and reduced myocardial scar size. Abundances of pro-angiogenic proteins were analyzed 12, 24 h and 1 month after the delivery of the regenerative substances. In a protein array, the significantly increased angiogenesis proteins were Activin A, Angiopoietin, Artemin, Endothelin-1, MCP-1; and remodeling factors ADAMTS1, FGFs, TGFb1, MMPs, and Serpins. In a qPCR analysis, increased levels of angiopeptin, CXCL12, HIF-1α and miR-132 were found 24 h after cell-based gene delivery, compared to those in untreated animals with infarction and in control animals. Expression of angiopeptin increased already 12 h after treatment, and miR-1 expression was reduced at that time point. In total, pMSC overexpressing HIF-1α showed beneficial effects for treatment of ischemic injury, mediated by stimulation of angiogenesis

Effect of Ischemic Preconditioning and Postconditioning on Exosome-Rich Fraction microRNA Levels, in Relation with Electrophysiological Parameters and Ventricular Arrhythmia in Experimental Closed-Chest Reperfused Myocardial Infarction

We investigated the antiarrhythmic effects of ischemic preconditioning (IPC) and postconditioning (PostC) by intracardiac electrocardiogram (ECG) and measured circulating microRNAs (miRs) that are related to cardiac conduction. Domestic pigs underwent 90-min. percutaneous occlusion of the mid left anterior coronary artery, followed by reperfusion. The animals were divided into three groups: acute myocardial infarction (AMI, n = 7), ischemic preconditioning-acute myocardial infarction (IPC-AMI) (n = 9), or AMI-PostC (n = 5). IPC was induced by three 5-min. episodes of repetitive ischemia/reperfusion cycles (rI/R) before AMI. PostC was induced by six 30-s rI/R immediately after induction of reperfusion 90 min after occlusion. Before the angiographic procedure, a NOGA endocardial mapping catheter was placed again the distal anterior ventricular endocardium to record the intracardiac electrogram (R-amplitude, ST-Elevation, ST-area under the curve (AUC), QRS width, and corrected QT time (QTc)) during the entire procedure. An arrhythmia score was calculated. Cardiac MRI was performed after one-month. IPC led to significantly lower ST-elevation, heart rate, and arrhythmia score during ischemia. PostC induced a rapid recovery of R-amplitude, decrease in QTc, and lower arrhythmia score during reperfusion. Slightly higher levels of miR-26 and miR-133 were observed in AMI compared to groups IPC-AMI and AMI-PostC. Significantly lower levels of miR-1, miR-208, and miR-328 were measured in the AMI-PostC group as compared to animals in group AMI and IPC-AMI. The arrhythmia score was not significantly associated with miRNA plasma levels. Cardiac MRI showed significantly smaller infarct size in the IPC-AMI group when compared to the AMI and AMI-PostC groups. Thus, IPC led to better left ventricular ejection fraction at one-month and it exerted antiarrhythmic effects during ischemia, whereas PostC exhibited antiarrhythmic properties after reperfusion, with significant downregulaton of ischemia-related miRNAs

Transcriptional Alterations by Ischaemic Postconditioning in a Pig Infarction Model: Impact on Microvascular Protection

Although the application of cardioprotective ischaemia/reperfusion (I/R) stimuli after myocardial infarction (MI) is a promising concept for salvaging the myocardium, translation to a clinical scenario has not fulfilled expectations. We have previously shown that in pigs, ischaemic postconditioning (IPostC) reduces myocardial oedema and microvascular obstruction (MVO), without influencing myocardial infarct size. In the present study, we analyzed the mechanisms underlying the IPostC-induced microvascular protection by transcriptomic analysis, followed by pathway analysis. Closed-chest reperfused MI was induced by 90 min percutaneous balloon occlusion of the left anterior descending coronary artery, followed by balloon deflation in anaesthetised pigs. Animals were randomised to IPostC (n = 8), MI (non-conditioned, n = 8), or Control (sham-operated, n = 4) groups. After three hours or three days follow-up, myocardial tissue samples were harvested and subjected to RNA-seq analysis. Although the transcriptome analysis revealed similar expression between IPostC and MI in transcripts involved in cardioprotective pathways, we identified gene expression changes responding to IPostC at the three days follow-up. Focal adhesion signaling, downregulated genes participating in cardiomyopathy and activation of blood cells may have critical consequences for microvascular protection. Specific analyses of the gene subsets enriched in the endothelium of the infarcted area, revealed strong deregulation of transcriptional functional clusters, DNA processing, replication and repair, cell proliferation, and focal adhesion, suggesting sustentative function in the endothelial cell layer protection and integrity. The spatial and time-dependent transcriptome analysis of porcine myocardium supports a protective effect of IPostC on coronary microvasculature post-MI

In vivo MRI and ex vivo histological assessment of the cardioprotection induced by ischemic preconditioning, postconditioning and remote conditioning in a closed-chest porcine model of reperfused acute myocardial infarction: importance of microvasculature

BACKGROUND: Cardioprotective value of ischemic post- (IPostC), remote (RIC) conditioning in acute myocardial infarction (AMI) is unclear in clinical trials. To evaluate cardioprotection, most translational animal studies and clinical trials utilize necrotic tissue referred to the area at risk (AAR) by magnetic resonance imaging (MRI). However, determination of AAR by MRI' may not be accurate, since MRI-indices of microvascular damage, i.e., myocardial edema and microvascular obstruction (MVO), may be affected by cardioprotection independently from myocardial necrosis. Therefore, we assessed the effect of IPostC, RIC conditioning and ischemic preconditioning (IPreC; positive control) on myocardial necrosis, edema and MVO in a clinically relevant, closed-chest pig model of AMI. METHODS AND RESULTS: Acute myocardial infarction was induced by a 90-min balloon occlusion of the left anterior descending coronary artery (LAD) in domestic juvenile female pigs. IPostC (6 x 30 s ischemia/reperfusion after 90-min occlusion) and RIC (4 x 5 min hind limb ischemia/reperfusion during 90-min LAD occlusion) did not reduce myocardial necrosis as assessed by late gadolinium enhancement 3 days after reperfusion and by ex vivo triphenyltetrazolium chloride staining 3 h after reperfusion, however, the positive control, IPreC (3 x 5 min ischemia/reperfusion before 90-min LAD occlusion) did. IPostC and RIC attenuated myocardial edema as measured by cardiac T2-weighted MRI 3 days after reperfusion, however, AAR measured by Evans blue staining was not different among groups, which confirms that myocardial edema is not a measure of AAR, IPostC and IPreC but not RIC decreased MVO. CONCLUSION: We conclude that IPostC and RIC interventions may protect the coronary microvasculature even without reducing myocardial necrosis

Mechanisms involved in ischemic conditioning of the heart evaluated in reperfused porcine model of myocardial infarction

Ischämische Konditionierung ist ein Konzept, bei dem kurze Episoden von sublethaler Ischämie/Reperfusion (I/R) vor oder nach einem Myokardinfarkt (MI) induziert werden, um Ischämie- und Reperfusionsschäden zu verringern. Diese kardioprotektive Konditionierung stimuliert Genexpressionsänderungen, posttranskriptionelle Vorgänge und parakrine- humorale Reaktionen, die zusammen das Herzgewebe gegen Ischämie schützen können. Obwohl die Konditionierung des Myokardiums eine viel versprechende Strategie gegen Ischämie/Reperfusionsschäden in Tierexperimenten ist, konnte eine klinische Verwendung keine zufriedenstellenden Resultate erreichen. Die primären Fragestellungen dieser Doktorarbeit waren (1) die Mobilisierungskapazität von CD34-positiven Zellen und die Sekretion von chemotaktischen Zytokinen im Großtiermodell im Schwein bei ischämischer Präkonditionierung (IPreC) und Postkonditionierung (IPostC) zu untersuchen; (2) den Effekt von IPostC auf die ventrikuläre Funktion und die Infarktgröße zu bestimmen; und (3) die Analyse des Transkriptoms von Myokardproben durchzuführen und die globale Genexpression zwischen IPostC und MI in Bezug auf zeitliche Änderungen und Region des Myokards zu vergleichen. Daher haben wir Konditionierungsimpulse vor und nach dem MI im translationalen Schweinemodell der akuten Myokardischämie mit anschließender Reperfusion bei geschlossenem Brustkorb eingesetzt. Die Tiere wurden in IPostC, IPreC, non-konditionierte MI und scheinoperierte Gruppen randomisiert. Die ventrikuläre Funktion wurde vor der Intervention, sowie nach drei Stunden und drei Tagen mit Echokardiographie untersucht und nach drei Tagen mit kardialer MRT evaluiert. Weiters wurden FACS Analysen zur Bestimmung der regenerativ CD34+ Zellzahl durchgeführt und die Konzentrationen von chemotaktischen Zytokinen bestimmt. Durch Verwendung von Next Generation Sequencing (NGS) Technologien haben wir die Auswirkung von IPostC auf die gesamte Genexpression im ischämischen und nicht-ischämischen (remote) Myokard im Schweinemodell untersucht. Wir konnten zeigen, dass IPreC die Sekretion von chemotaktischen Zytokinen und die Auswanderung von CD34+ Zellen steigert. Im Gegensatz dazu hatte IPostC keinen Einfluss auf die Migration von blutbildenden Zellen. Wir konnten zeigen, dass die Regulation der MMP-2 Sekretion durch IPreC invers reguliert wird, und vermehrt zu anti-ischemischen Effekten beitragen kann. Im Vergleich zu unbehandeltem MI, reduzierte IPostC unabhängig von der Infarktgröße das myokardiale Ödem und die mikrovaskuläre Obstruktion. Die Analyse des Transkriptoms zeigte, dass die Komponenten des “p o-survival kinas ” Signa w gs, Phosphoinositid-3-Kinasen- Akt Signalwegs und „survival activating factor enhancement“ Signalwegs in MI und IPostC in beiden Zeitfenstern und myocardialen Zonen ähnlich reguliert sind. Die weitere bioinformatische Analyse ergab nach drei Tagen in der IPostC Gruppe eine signifikante Überexpression von Genen, die an der Regulation der fokalen Adhäsion beteiligt sind und die down-regulierte Expression von Transkripten, die in der Aktivierung von Blutzellen, sowie in Hypertrophie und Kardiomyopathie involviert sind. All dies deutet darauf hin, dass die IPostC auf mikrovaskulärer Ebene wirkt. Unsere Ergebnisse liefern Beweise für die Kapazität der IPreC eine humorale Reaktion zu stimulieren. Die erhaltenen funktionellen Daten und Analysen des Transkriptoms zeigen eine potenzielle Kardioprotektion der IPostC in Bezug auf einen positiven Effekt ausschließlich auf Ebene der Mikrogefäße.Transient episodes of sublethal ischemia/reperfusion (I/R) represent conditioning stimuli aiming to render the myocardium more resistant against subsequent ischemia/reperfusion (I/R) injury. Stimulation of cardioprotective processes at the level of gene expression, protein post-transcriptional modifications, and paracrine-humoral responses may synergistically protect the tissue subjected to ischemia. Although the concept of ischemic conditioning showed a promising strategy to attenuate reperfusion-induced injury in pre-clinical animal models, translation to clinical scenarios has not met with much success. The primary aims of this doctoral thesis were: (1) to assess whether ischemic preconditioning (IPreC) or postconditioning (IPostC) stimuli induce mobilisation of regenerative CD34+ cells and the release of chemotactic cytokines in a clinically relevant porcine model of reperfused myocardial infarction (MI); (2) to evaluate the effect of IPostC on left ventricular (LV) function and infarct size; and (3) to analyse transcriptome of the myocardium affected by IPostC and to compare transcriptional pattern with the MI model, particularly with respect to the time window and myocardial regions. To achieve these aims, we applied conditioning stimuli prior to, or after, prolonged MI in closed-chest reperfused swine models in IPostC and IPreC groups, respectively. Animals were randomised in the IPostC, IPreC, non-conditioned MI and sham-operated control groups. We assessed LV function at the baseline, three hours and three days follow-up by echocardiography and at three days follow-up by cardiac MRI. To address the cell mobilisation in IPreC and IPostC we measured the absolute cell count of CD34+ cells by FACS analysis and evaluated the release of associated chemotactic cytokines. Applying the next generation sequencing approach, we analysed the gene expression changes in the transcriptome of the IPostC and MI models. Our group has shown that IPreC, but not IPostC, induces the release of chemotactic cytokines and egression of CD34+ hematopoietic cells into peripheral circulation in the late phase of cardioprotection. We found opposite regulation of MMP-2 plasma release in IPostC and IPreC, which may contribute to the cardioprotective effect of IPreC. Compared to MI, IPostC was associated with protection on a microvascular level with attenuated oedema and microvascular obstruction independently from changes in infarct size. Overall, the expression of genes involved in pro-survival kinase pathway, phosphatidylinositol 3-kinase-Akt signalling, and survivor activating factor enhancement pathways were mostly similar in IPostC and MI experimental groups in both time windows and tissue regions. However, we observed significant deregulation of gene sets enriched in focal adhesion pathway in the late window of cardioprotection after IPostC. Transcripts involved in adhesion and activation of blood cells, cardiac hypertrophy and cardiomyopathy were downregulated, indicating a beneficial effect of IPostC on microvasculature. Thus, using a swine model of MI, we have demonstrated that mobilisation of CD34+cells and release of chemotactic cytokines contributes to IPreC, but not IPostC, cardioprotection. The data obtained by cMRI measurements and transcriptome analysis suggests that the potential cardioprotective effect of IPostC is limited to changes on a microvascular level.submitted by MSc Dominika LudovicZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität, Dissertation, 2016OeB

Alternative Splicing in Cardiovascular Disease—A Survey of Recent Findings

Alternative splicing, a driver of posttranscriptional variance, differs from canonical splicing by arranging the introns and exons of an immature pre-mRNA transcript in a multitude of different ways. Although alternative splicing was discovered almost half a century ago, estimates of the proportion of genes that undergo alternative splicing have risen drastically over the last two decades. Deep sequencing methods and novel bioinformatic algorithms have led to new insights into the prevalence of spliced variants, tissue-specific splicing patterns and the significance of alternative splicing in development and disease. Thus far, the role of alternative splicing has been uncovered in areas ranging from heart development, the response to myocardial infarction to cardiac structural disease. Circular RNAs, a product of alternative back-splicing, were initially discovered in 1976, but landmark publications have only recently identified their regulatory role, tissue-specific expression, and transcriptomic abundance, spurring a renewed interest in the topic. The aim of this review is to provide a brief insight into some of the available findings on the role of alternative splicing in cardiovascular disease, with a focus on atherosclerosis, myocardial infarction, heart failure, dilated cardiomyopathy and circular RNAs in myocardial infarction

Human recombinant activated protein C-coated stent for the prevention of restenosis in porcine coronary arteries

Activated protein C (APC), an endogenous protein, inhibits inflammation and thrombosis and interrupts the coagulation cascade. Here, we investigated the effect of human recombinant APC on the development of neointimal hyperplasia in porcine coronary arteries. Yukon Choice bare metal stents were coated with 2.6 mu g APC/mm(2). Under general anesthesia, APC-coated and bare stents were implanted in the left anterior descending and circumflex coronary arteries of 10 domestic pigs. During the 4-week follow-up, animals were treated with dual antiplatelet therapy and neointimal hyperplasia was evaluated via histology. Scanning electron microscopy indicated successful but unequal coating of stents with APC; nearly complete drug release occurred within 4 h. Enzyme-linked immunosorbent assay revealed that intracoronary stent implantation rapidly increased the levels of monocyte chemoattractant protein-1, an effect that was inhibited by APC release from the coated stent. Fibrin deposition and adventitial inflammation were significantly decreased 1 month after implanting APC-coated stents versus bare stents, paralleled by significantly smaller neointimal area (0.98 +/- 0.92 vs. 1.44 +/- 0.91 mm(2), P = 0.028), higher lumen area (3.47 +/- 0.94 vs. 3.06 +/- 0.91 mm(2), P = 0.046), and lower stenosis area (22.2 +/- 21.2 % vs. 32.1 +/- 20.1 %, P = 0.034). Endothelialization was complete with APC-coated but not bare (90 %) stents. P-selectin immunostaining revealed significantly fewer activated endothelial cells in the neointima in the APC group (4.6 +/- 1.9 vs. 11.6 +/- 4.1 %, P < 0.001). Thus, short exposure of coronary arteries to APC reduced inflammatory responses, neointimal proliferation, and in-stent restenosis, offering a promising therapy to improve clinical outcomes of coronary stenting. However, coating stents with APC for prolonged, controlled drug release remains technically challenging

MiR-21, MiR-29a, GATA4, and MEF2c Expression Changes in Endothelin-1 and Angiotensin II Cardiac Hypertrophy Stimulated Isl-1+Sca-1+c-kit+ Porcine Cardiac Progenitor Cells In Vitro

Cost- and time-intensive porcine translational disease models offer great opportunities to test drugs and therapies for pathological cardiac hypertrophy and can be supported by porcine cell culture models that provide further insights into basic disease mechanisms. Cardiac progenitor cells (CPCs) residing in the adult heart have been shown to differentiate in vitro into cardiomyocytes and could contribute to cardiac regeneration. Therefore, it is important to evaluate their changes on the cellular level caused by disease. We successfully isolated Isl1+Sca1+cKit+ porcine CPCs (pCPCs) from pig hearts and stimulated them with endothelin-1 (ET-1) and angiotensin II (Ang II) in vitro. We also performed a cardiac reprogramming transfection and tested the same conditions. Our results show that undifferentiated Isl1+Sca1+cKit+ pCPCs were significantly upregulated in GATA4, MEF2c, and miR-29a gene expressions and in BNP and MCP-1 protein expressions with Ang II stimulation, but they showed no significant changes in miR-29a and MCP-1 when stimulated with ET-1. Differentiated Isl1+Sca1+cKit+ pCPCs exhibited significantly higher levels of MEF2c, GATA4, miR-29a, and miR-21 as well as Cx43 and BNP with Ang II stimulation. pMx-MGT-transfected Isl1+Sca1+cKit+ pCPCs showed significant elevations in MEF2c, GATA4, and BNP expressions when stimulated with ET-1. Our model demonstrates that in vitro stimulation leads to successful Isl1+Sca1+cKit+ pCPC hypertrophy with upregulation of cardiac remodeling associated genes and profibrotic miRNAs and offers great possibilities for further investigations of disease mechanisms and treatment