On endovascular transplantation of modified mRNA enhanced cells and treatment of cardiac arrest using levosimendan : leveraging modRNA therapeutics and cardioprotective pharmacology to improve cardiovascular outcomes

<jats:p>&lt;p dir="ltr"&gt;Background: Ischaemic cardiovascular disease causes tissue injury through both acute perfusion failure and insufficient repair. In the heart, restoring blood flow is essential, but it does not fully define whether injured myocardium recovers, progresses to scar, or contributes to cardiac arrest and organ failure. Effective therapy therefore depends on the therapeutic agent, the timing of treatment, the delivery route, and the physiological state of the injured tissue. This thesis aimed to investigate targeted endovascular delivery of modified mRNA-enhanced human mesenchymal stromal cells, levosimendan during ischaemic cardiac arrest, and early myocardial tissue changes during ischaemia and reperfusion.&lt;/p&gt;&lt;p dir="ltr"&gt;Methods: We electroporated human mesenchymal stromal cells with modified mRNA encoding vascular endothelial growth factor A and evaluated them in vitro and after trans-vessel wall endovascular delivery to heart and kidney in large- animal models, using either the Extroducer or a 26G mock-up device. We compared cell biodistribution and retention after intravenous, intra-arterial, and targeted trans-vessel wall delivery using positron emission tomography with radiolabelled cells, and quantified protein by ELISA. We evaluated levosimendan during cardiopulmonary resuscitation in a swine model of ischaemic cardiac arrest. In a separate large-animal model, we performed multiparametric cardiac magnetic resonance imaging during coronary occlusion and during the first minutes after reperfusion to assess perfusion, regional function, T1 and T2 changes, and final infarction by triphenyltetrazolium chloride staining.&lt;/p&gt;&lt;p dir="ltr"&gt;Results: Modified mRNA-enhanced human mesenchymal stromal cells retained viability after electroporation and catheter passage and produced high local vascular endothelial growth factor A levels after myocardial delivery. At 24 hours, myocardial vascular endothelial growth factor A was 41.1 + 15.7 μg/mL/g tissue after delivery of modified mRNA-enhanced cells, compared with 0.147 + 0.03 μg/mL/g after naïve cells and 0.127 + 0.09 μg/mL/g after modified mRNA in buffer. Targeted trans-vessel wall delivery increased local cell retention compared with conventional routes. In the heart, median retention was 38% after Extroducer delivery, compared with 5% after the 26G mock-up device, 6% after intra-arterial delivery, and 0.1% after intravenous delivery. In the kidney, corresponding values were 14%, 3%, 5%, and 0.12%. Intra-arterial delivery produced broader organ distribution but limited parenchymal deposition, whereas targeted delivery produced focal cell clusters at injection sites and higher local protein expression.&lt;/p&gt;&lt;p dir="ltr"&gt;During ischaemic cardiac arrest, levosimendan improved resuscitation physiology without compromising systemic arterial pressure. Levosimendan lowered pulmonary arterial pressure, reduced central venous pressure, improved gas exchange, reduced adrenaline requirement after return of spontaneous circulation, and increased the probability of return of spontaneous circulation compared with placebo in a dose-dependent manner.&lt;/p&gt;&lt;p dir="ltr"&gt;Multiparametric cardiac magnetic resonance imaging showed that myocardial injury markers were already present during coronary occlusion. Perfusion in the area at risk increased rapidly after reflow but remained lower than in remote myocardium. Native T1-derived area at risk showed strong agreement with perfusion-defined area at risk, while T2 also correlated with perfusion but with greater variability. Regional myocardial function remained impaired despite improved perfusion, and magnetic resonance imaging-derived area at risk generally exceeded final infarct size.&lt;/p&gt;&lt;p dir="ltr"&gt;Conclusion: Across the ischaemic arc, cardiovascular interventions cannot be judged by the therapeutic agent alone. This thesis shows that modified mRNA- enhanced cells raise local protein production by more than two orders of magnitude over naïve cells or modified mRNA alone, but only where delivery retains them within the target parenchyma, that levosimendan may improve resuscitation through effects on right-sided and pulmonary vascular physiology, and that myocardial injury begins before reperfusion and continues to evolve through the first minutes after flow is restored. What is delivered, how it is delivered, and when it is delivered are therefore not separable questions. Translating regenerative and cardioprotective strategies towards clinical use will require answering all three.&lt;/p&gt;&lt;h3 dir="ltr"&gt;List of scientific papers&lt;/h3&gt;&lt;p dir="ltr"&gt;The thesis includes the following articles, which are referred to in the text by Roman numerals&lt;/p&gt;&lt;p dir="ltr"&gt;I. &lt;b&gt;Al-Saadi J,&lt;/b&gt; Walden M, Sandell M, Sohlmér J, Grankvist R, Friberger I, Andersson A, Carlsten M, Chien K, Lundberg J, Witman N, Holmin S. Endovascular transplantation of mRNA-enhanced mesenchymal stromal cells results in superior therapeutic protein expression in swine heart. Molecular Therapy: Methods &amp; Clinical Development. 2024;32:101225. &lt;a href="https://doi.org/10.1016/j.omtm.2024.101225"&gt;https://doi.org/10.1016/j.omtm.2024.101225&lt;br&gt;&lt;/a&gt;&lt;br&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;II. &lt;b&gt;Al-Saadi J,&lt;/b&gt; Walden M, Lövljung V, Witman N, Sohlmér J, Grankvist R, Sandell M, Chien K, Goos J, Lundberg J, Holmin S. Endovascular Targeted Parenchymal Delivery with Extroducer Changes Distribution and Significantly Enhances Cell Retention Compared to Other Delivery Methods. [Manuscript]&lt;/p&gt;&lt;p dir="ltr"&gt;III. Rysz S, &lt;b&gt;Al-Saadi J,&lt;/b&gt; Campoccia Jalde F, Nyman J, Lundberg J, Jonsson Fagerlund M, Djärv T. Levosimendan improves central haemodynamic status and gas exchange in a model of ischaemic cardiac arrest: A large animal study. European Journal of Anaesthesiology. 2025;42:518-526. &lt;a href="https://doi.org/10.1097/EJA.0000000000002144"&gt;https://doi.org/10.1097/EJA.0000000000002144&lt;br&gt;&lt;/a&gt;&lt;br&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;IV. &lt;b&gt;Al-Saadi J,&lt;/b&gt; Rysz S, Djärv T, Lange C, Sörensson P, Carlsson M, Jonsson-Fagerlund M, Lundberg J. Multiparametric MRI Characterization of Early Tissue Injury Before and Immediately After Reperfusion in Acute Myocardial Infarction. [Manuscript]&lt;/p&gt;</jats:p>