RNA interference (RNAi) is a conservative post-transcriptional gene silencing mechanism that can be mediated by small interfering RNAs (siRNAs). Given the effectiveness and specificity of RNAi, the administration of siRNA molecules is a promising approach to cure diseases caused by abnormal gene expression. However, as siRNA is susceptible to degradation by nucleases and it can hardly penetrate cell membranes due to its polyanionic nature, a successful translation of the RNAi mechanism for therapeutic purposes is contingent on the development of safe and efficient delivery systems. This dissertation described the development of novel siRNA delivery systems on the basis of polymeric and dendrimeric materials and also demonstrated the application of one optimized delivery system to deliver therapeutic siRNAs in a cardiovascular disease model in vivo. We studied a linear peptide polymer made from cell penetrating peptide monomers and investigated the contribution of the polymeric structure, degradability, and ligand conjugation to the siRNA loading capacity, biocompatibility, and transfection efficiency of polymeric materials. With the obtained knowledge and experience, we invented a neutral crosslinked delivery system aiming to solve the inherent drawbacks of traditional cationic delivery systems that are based on electrostatic interactions. The new concept utilized buffering amines to temporarily bind siRNA and a crosslinking reaction to immobilize the formed particles, and targeting ligands modified on the neutral dendrimer surface further enhanced the interactions between the delivery vehicles and target cells. The obtained delivery system allowed stability, safety, controllability, and targeting ability for siRNA delivery, and the method developed here could be transformed to other polymeric or dendrimeric cationic materials to make them safer and more efficient. To exploit the therapeutic potential of siRNA delivery, we developed a tadpole-shaped dendrimeric material to deliver siRNA against an Angiotensin II receptor in a rat ischemia-reperfusion model. Our results showed that the nonaarginine-conjugated tadpole dendrimer was capable of delivering siRNA effectively to cardiac cells both in vitro and in vivo, and the successful down-regulation of the Angiotensin II receptor preserved the cardiac functions and reduced the infarct size post-myocardial infarction. This dissertation paves a way for transforming multifunctional non-viral siRNA delivery systems into potent therapeutic strategies for the management of cardiovascular diseases.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Heart failure (HF) following myocardial infarction (MI) is the leading cause of mortality and morbidity worldwide. Existing medical and interventional therapies can only reduce the cardiomyocytes (CMs) lost during MI. They are unable to replenish the permanent loss of CMs and this contributes to progressive pathological left ventricular (LV) remodeling and HF. Cell-based therapies using adult stem cells or embryonic stem cells (ESCs) and their cardiac derivatives have frequently been explored as a potential therapeutic approach to restore cardiac function in HF. The objectives of this thesis are to evaluate the efficacy and safety of different approaches of stem cell based therapy to improve cardiac function using small and large animal MI models. In Chapter 3, we studied the functional consequences of direct intramyocardial transplantation of ESCs and ESC-derived cardiomyocytes (ESC-CMs) in a murine model of acute MI. LV ejection fraction (LVEF) and maximal positive or negative pressure derivative (dP/dt) improved 4 weeks after transplantation of either ESCs or ESC-CMs. Nevertheless there was a higher incidence of inducible ventricular tachyarrhythmia (VT) and higher mortality in animals transplanted with ESC-CMs than those with ESCs. At a single cell level, ESC-CMs exhibited immature electrophysiological properties such as depolarized resting membrane potential (RMP), longer action potential duration (APD) and automaticity. In Chapter 4, we tested the hypothesis that genetic modification of these immature electrophysiological properties of ESC-CMs by overexpression of Kir2.1 gene encoding the ion channels for IK1, may alleviate the pro-arrhythmic risk. In this study, Kir2.1 channels expression could be controlled with the administration of doxycycline (DOX). The DOX-treated ESC-CMs were more mature with hyperpolarized RMP and shorter APD than their counterparts without DOX treatment. A similar improvement in LV systolic function was observed 4 weeks after both DOX treated and untreated ESCCMs transplantation, although those animals transplanted with DOX-treated ESC-CMs had a significantly lower incidence of spontaneous and inducible VT. Histological analysis in both studies suggested that the major mechanisms of improvement in cardiac function were related to angiogenesis and low apoptosis rate of native cardiomyocytes mediated via paracrine effects. Importantly, very limited retention of ESC-CMs was observed 4 weeks after transplantation. Cell-based patches that use different bioengineering techniques have been proposed to improve cell retention and survival following transplantation. In Chapter 5, the efficacy of a passive epicardial patch was tested in a chronic large animal MI model with HF created with catheter-based coronary embolization. The implantation of an epicardical patch over the infarcted LV region was performed 8 weeks after MI in pigs with impaired LVEF. At week 20, pigs implanted with epicardical patches had significantly thicker LV wall thickness at the infarction sites, smaller LV dilation and better LV systolic function compared with control animals. The expression of MMP-9 was significant lower in the epicardical patch group at the peri-infarct zones. These findings suggested that a passive epicardial patch can improve LV function in HF and provides important proof-of-principle data to support its use as a platform for delivery of cell-based therapies after MI. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Remifentanil is an ultra-short-acting phenylpiperidine opioid analgesic that is rapidly metabolized by nonspecific blood and tissue esterases. In clinical practice, remifentanil is now more commonly used during both cardiac and non-cardiac surgery than classic opioid agonists such as morphine, since it can be given in higher doses, is more titratable and enables fast recovery of patients in the postoperative period. Remifentanil preconditioning (RPC), achieved by intravenous remifentanil infusion interspersed with infusion-free periods before indexed ischaemia, attenuates cardiac ischaemia-reperfusion injury (IRI). This is experimentally manifested by reduced postischaemic myocardial infarct size (IS) and diminished markers of cardiac failure and apoptosis, and, clinically, by reduced release of biomarkers of myocardial cellular injury after cardiac surgery. However, the underlying mechanisms by which RPC has a cardioprotective effect need to be further explored. It’s generally considered that the Reperfusion Injury Salvage Kinase (RISK) pathway, triggering the expression of phosphatidylinositol 3-kinase (PI3K) as well as Akt, exerts a pivotal role in both classic ischaemic preconditioning (IPC) and pharmacological preconditioning induced cardioprotection. Moreover, recent studies show that the Survivor Activating Factor Enhancement (SAFE) signalling pathway, which involves signal transducers and activators of transcription-3 (STAT3) and janus activated kinase-2 (JAK2), also has an essential role in IPC. Although cross-talk has been found between the RISK and SAFE pathways, the SAFE pathway can function independently of the RISK to confer cardioprotection. However, the roles of JAK/STAT and PI3K/Akt signalling and, in particular, their relative importance in RPC-mediated cardioprotection have not been studied. I explored whether RPC confers cardioprotection via the JAK/STAT or PI3K/Akt pathway and its relationship with GSK3β inhibition. In first part of my study, I explored relative role of the JAK/STAT and PI3K/Akt which were involved in RPC cardioprotection using JAK2 and PI3K inhibition. Male Sprague-Dawley rats were either sham operated or randomly assigned to receive I/R alone or as well as RPC. Pretreatment with the JAK2 inhibitor AG490 or the PI3K inhibitor wortmannin was induced before ischaemia in rats. RPC reduced myocardial infarction and haemodynamic dysfunction induced by IRI accompanied with increased phosphorylation of STAT3 but not Akt or eNOS phosphorylation. AG490 but not wortmannin cancelled RPC’s cardioprotection. In addition, RPC attenuated hypoxia/reoxygenation induced cardiomyocyte apoptosis while STAT3 knock-out abolished the protective effects of RPC. These findings suggest that RPC confers cardioprotection primarily via activation of the JAK/STAT signalling but not the PI3K/Akt signalling pathway. The second study further investigated the role of GSK3β in RPC cardioprotection using the GSK3β inhibitor SB216763. I found that SB restored the ability of RPC to reduce the extent of myocardial infarction and CK-MB release despite the presence of AG490. The phosphorylation of GSK3β was increased by RPC. In addition, GSK3β gene knock-out with siRNA preserved RPC’s cardioprotection regardless of STAT3 abrogation indicating that GSK3β inhibition plays a critical role as a downstream effector in RPC mediated cardioprotection. Taken together with the evidence from this two part study, I conclude that RPC confers cardioprotection by activating the JAK/STAT and, subsequently, inhibiting GSK3β, a critical downstream effector of RPC cardioprotection. / published_or_final_version / Anaesthesiology / Master / Master of Philosophy

In 2015, American Heart Association (AHA) reported that 1 in 9 deaths are attributed to Heart failure (HF), the number one killer in the world. While advancements in interventional cardiology in conjunction with pharmacotherapies have significantly reduced the rate of mortality following MI, there has been a corresponding rise in chronic heart failure (CHF) in surviving patients, largely attributed to the limited regenerative capacity of the heart and the inadequate healing response. Myocardial ischemic injury triggers an exuberant local and systemic inflammation, and the extent and quality of the cardiac wound healing process is intricately tied to the delicate equilibrium of this inflammatory response. While cardiac regeneration is an important goal, it is imperative in the meantime to explore therapeutic strategies that target these inflammatory mediators of early cardiac repair. These interventions to influence and improve cardiac wound healing can represent a new therapeutic window to halt the progression of heart failure between the few hours that may be used to limit infarct size by reperfusion and an irreversible non-contractile cardiac scar. This dissertation examines three therapeutic delivery strategies aimed at modulating the immune response to enhance cardiac repair in rodent models MI: 1) Polyketal nanoparticles as siRNA delivery vehicles for antioxidant therapy; 2) Spherical nucleic acid particles for anti-inflammatory therapy and; 3) Bioactive PEG (polyethyleneglycol)-based hydrogel for immunomodulation. The work presented here applies novel nucleic acid delivery strategies for cardiac gene silencing and has contributed to new knowledge with regard to modulating the immune response following MI.

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Σκοπός: Ο σκοπός της παρούσας μελέτης ήταν να ερευνηθεί κατά πόσο η ανάσπαση του τμήματος ST και η θετικοποίηση του κύματος Τ στις Q – απαγωγές του ΗΚΓ κατά τη διάρκεια της δοκιμασίας κόπωσης πρό της εξόδου από το νοσοκομείο, μπορούν να συνεισφέρουν στη κλινική αντιμετώπιση των ασθενών με πρόσφατο έμφραγμα μυοκαρδίου. Εισαγωγή: Η κλινική σημασία αυτών των ΗΚΓ ευρημάτων παραμένει αμφισβητούμενη, παρά το γεγονός ότι σε αρκετές μελέτες σχετίζεται με την ύπαρξη μυοκαρδιακής βιωσιμότητας όπως και λειτουργικής αποκατάστασης μετά από επαναγγείωση. Από τη στιγμή που η επιλογή των ασθενών που συμμετέχουν σε κάθε μελέτη μπορεί να εξηγήσει τα διαφορετικά αποτελέσματα, η αξία αυτών των ευρημάτων στη τωρινή εποχή της θρομβόλυσης πρέπει να διευκρινισθεί. Μέθοδος: Στη μελέτη έλαβαν μέρος 101 ασθενείς, ηλικίας 58+11 έτη, με πρόσφατο, πρώτο, ανεπίπλεκτο Q – έμφραγμα μυοκαρδίου (εκ των οποίων 56% ήταν πρόσθια, 57% θρομβολυμένα με κλάσμα εξώθησης αριστεράς κοιλίας 43 + 7%). Οι ασθενείς προ της εξόδου από το νοσοκομείο υποβάλλονταν σε υπομεγίστη δοκιμασία κόπωσης. Ακολούθως, σε απουσία σοβαρής ισχαιμίας, διενεργείτο διαδοχικά μελέτη δυναμικής ηχοκαρδιογραφίας με χρήση δοβουταμίνης (DSE), σπινθηρογράφημα μυοκαρδίου με θάλλιο – 201 (201Tl-SPECT) και αγγειογραφία των στεφανιαίων αρτηριών. Αποτελέσματα: Οι ασθενείς με ανάσπαση του τμήματος ST στη μέγιστη κόπωση έχουν μεγαλύτερης έκτασης εμφράγματα (peak CPK 2351+1465 versus 1671+1132 U/L, p<0.05) και περισσότερο επηρεασμένη συστολική λειτουργία με κριτήριο τον αριθμό των μυοκαρδιακών τμημάτων με συμπεριφορά ουλώδους ιστού τόσο στη δυναμική ηχοκαρδιογραφία με χρήση δοβουταμίνης (p<0.05) όσο και στο σπινθηρογράφημα μυοκαρδίου με θάλλιο – 201 (p<0.01). Η επίπτωση βιωσιμότητας και / ή ισχαιμίας δεν διέφερε στις δύο ομάδες. Τα αποτελέσματα ήταν παρόμοια και στη χαμηλής φόρτισης κόπωση. Η εντόπιση του εμφράγματος στο πρόσθιο τοίχωμα και η παρουσία > 3 τμημάτων με συμπεριφορά ουλής στη DSE ήταν μεταξύ των ανεξάρτητων θετικών προβλεπτικών παραγόντων της ανάσπασης του ST στη μέγιστη άσκηση. Η θετικοποίηση του κύματος Τ απεδείχθει το ίδιο αναποτελεσματική στη πρόβλεψη βιωσιμότητας και / ή ισχαιμίας. Κατά τη διάρκεια της μακρόχρονης κλινικής παρακολούθησης που ακολούθησε (31+13 μήνες), η συχνότητα των συμβαμάτων ήταν χαμηλή (8 % για θάνατο ή μη θανατηφόρο ΟΕΜ) και δεν διέφερε μεταξύ των ομάδων. Συμπέρασμα: Σε ασθενείς με πρόσφατο Q – έμφραγμα μυοκαρδίου, χωρίς κλινικά και ΗΚΓ κριτήρια σημαντικής υπολειπομένης ισχαιμίας, οι ανασπάσεις του διαστήματος ST κατά την δοκιμασία κοπώσεως εκφράζουν την μεγάλη έκταση του εμφράγματος. Η δυνατότητα αυτών των ST/T μεταβολών του ΗΚΓ να συνεισφέρουν στη ανίχνευση βιωσιμότητας και ισχαιμίας και στη διαστρωμάτωση κινδύνου αυτών των ασθενών , αποδεικνύεται χαμηλή. Τά κλασσικά κλινικά κριτήρια που χρησιμοποιούνται και σήμερα αποδεικνύονται ιδιάτερα αποτελεσματικά στη επιλογή των ασθενών χαμηλού κινδύνου. / Objectives: The aim of this prospective study was to investigate whether ST segment elevation and T wave normalization in Q-wave leads on pre-discharge exercise electrocardiogram (ECG) can contribute to patient management after recent myocardial infarction (MI) Background: The clinical relevance of these exercise ECG changes remains controversial despite accumulating evidence of their association with myocardial viability as well as functional recovery after revascularization. Since patient selection may explain the discordant results across the studies, the value of these ST/T abnormalities in the thrombolytic era should be better defined. Methods: One-hundred one patients, aged 58+11 years, with a recent, first, uncomplicated Q-wave MI (56% anterior, 57% thrombolyzed, ejection fraction 43+7%) underwent predischarge, submaximal treadmill testing followed, in the absence of severe ischemia, by dobutamine stress echocardiography, thallium-201 single photon emission computed tomography and coronary angiography. Results: Patients with, as compared with those without ST elevation at peak exercise had more severe infarctions (peak creatine kinase 2351+1465 versus 1671+1132 U/L, p<0.05) and more extensively impaired left ventricular contractility due to scar tissue as based on the number of segments with scar on echocardiography (p<0.05) or scintigraphy (p<0.01). However, the incidence of myocardial viability and ischemia was not different between the two groups. Results were similar for ST elevation at low level exercise. Anterior infarction location and at least three scarred segments on dobutamine stress echocardiography were among the independent predictors of ST elevation at peak ergometric exercise. T wave normalization was similarly inaccurate in identifying viability or ischemia. Over long-term follow-up of 31+13 months, the event rate was low ( 8 % for death or nonfatal MI) and did not differ between groups with or without these exercise-induced ST/T wave changes. Conclusions: In patients after acute, Q-wave MI without severe ischemia according to clinical and standard ECG criteria, exercise-induced ST elevation is associated with larger infarctions. The contribution of these ST/T changes to identify patients with myocardial viability or ischemia and for risk stratification is poor. In-hospital management of these patients based on routine clinical practice is sufficient for selection of a population with a relatively low long-term risk.

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