Tese de Doutoramento Medicina 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

In the last three decades, remarkable improvements in patient care, namely a widened access to reperfusion therapy, have contributed decisively to a consistent decrease in acute myocardial infarction-related mortality and other health outcomes. However, survivors with residual systolic dysfunction and symptomatic heart failure still pose significant challenges. Several treatment options, such as reperfusion-adjuvant drugs, targeting patients at high risk of sustaining LV dysfunction despite otherwise optimal guideline-directed therapy, have failed to deliver consistent results. The extremely complex pathophysiological mechanisms underlying both ischemic and reperfusion injury are addressed. Aside from the thrombotic component of coronary occlusion, a highly regulatedimmune and inflammatory response orchestrated by local and migrating cells and mediated by soluble mediators (such as cytokines, growth factors, chemokines, mRNAs and many other molecules) plays a key role in infarct extension and healing. The substantial individual variability in the way these mechanisms operate and interact with each-other to produce the final consequences of acute myocardial infarction on left ventricular function, and on clinical outcomes, is highlighted. Elucidating and understanding this intricate network has become an important contribution of the recent developments in cell and regenerative therapy and has been crucial to the comprehension of its mechanistic effects, as well as for the development of new therapeutic targets. Considering the vastness of published literature, a choice was made to undertake a concise review, targeting experimental evidence on cellular and molecular signalling pathways for which an effect of exogenous cells has been suggested or demonstrated. Cell-based therapies have been initially developed and emerged as a strategy for reversing scar tissue and recovering left ventricular function, under the assumption that, once delivered to the heart, exogenous cells would engraft and replace lost cells by transdifferentiating into the full set of myocardial cellular components. This concept, however, soon failed to hold true, at least as the primary mechanism underlaying the biological effects of cell therapy. Instead, continued investigation has made clear that exogenous cells exert their effects mostly through modulation of inflammation, apoptosis, angiogenesis, recruitment of endogenous precursor cells and cell reprograming, as part of an intricate process that is mostly mediated by paracrine secretion of many distinct types of signalling molecules, commonly designated as secretome. This secretory profile (considering both the type of secreted molecules and their functional potency) is not identical in all cell types and can decisively influence biological effects. Although many cell types have been used in both experimental and clinical studies, autologous myoblasts and, subsequently, bone marrow-derived cells (whole BMC or some ubfraction) have been the most widely investigated. However, despite early enthusiastic results both in animal disease models and human studies, evidence from rigorously conducted clinical trials has been less compelling, despite aggregate data suggests benefits on LV remodelling, ejection fraction, and reduced heart failure-related adverse health outcomes. Reasons for such discrepancy stem from several intertwined biological and methodological factors, including, as mentioned, a misleading theoretical framework. In the majority of studies, including large animal models and human trials, cells were delivered into the heart when most of the cellular and molecular processes leading to scar formation and remodelling had already occurred, or were in an advanced stage. Mesenchymal stromal (or stem) cells (MSC) are multi-lineage precursor cells capable of immunemodulation and secretion of molecules known to regulate inflammation an interfere with many of the pathways involved in tissue reparation and regeneration. These cells can be easily obtained from widely available human autologous and allogeneic sources, the umbilical cord being the most abundant. In particular, human umbilical cord matrix-derived mesenchymal stromal cells (hUCM-MSC) can be safely expanded to large amounts, have enhanced functional secretory capacity relative to other types of adult precursor cells, have shown effectiveness in animal studies, and safety in phase I-II human trials of myocardial infarction. In contrast to most prior experimental studies, we investigated the concept of early intra-coronary injection of hUCM-MSC as reperfusion-adjuvant therapy, using a large-animal pre-clinical model of AMI with reperfusion, and a randomized placebo-controlled and blinded trial design. A fully percutaneous model of myocardial infarction with reperfusion was successfully created in swine, allowing an experimental setup that is much closer to actual clinical practice - in which most patients sustaining an AMI will receive reperfusion - as opposed to the classical open-chest and permanent vessel ligation models. Efficacy was evaluated using several validated methodologies. Mechanical and functional outcomes consisted of invasive pressure-volume loop analysis with high-fidelity conductance catheters and morphometric echocardiography. Mechanistic readouts included histological infarct size, fibrosis and hypertrophy in the remote areas, strength-length relationship studies in isolated skinned cardiomyocytes, differential gene expression by transcriptome sequencing analysis by RNA-seq and determination of serum biomarkers (such as galectin-3). It was found that, as compared to placebo, intra-coronary transfer of hUCM-MSC was associated with significant improvements in PV loop-derived left ventricular systolic function parameters, which appeared to be notoriously independent from reduction in infarct size. In addition, interstitial fibrosis and compensatory cardiomyocyte hypertrophy were attenuated in tissue biopsies from the remote myocardium of animals treated with hUCM-MSC, which was consistent with differential expression of genes related to adverse matrix remodelling and lower plasma levels of galectin-3. Taken together, these results suggest that the putative benefits of treatment with hUCM-MSC as reperfusion-adjuvant therapy, may be mediated by modulation of the compensatory response of the remaining myocardium to the ischemic injury, involving mechanisms that seem to be unrelated to the generation of new contractile cardiomyocytes or extensive reductions in infarct size. This is in line with most recent evidence concerning the biological effects of cell-based treatments, supporting the shift in the initial theoretical construct underlying cell-therapy. Also, they contribute to reinforce the potential of hUCM-MSC as adjuvant therapy for myocardial infarction and sustain the need for further experimentation and well-designed clinical studies. Finally, the current status of stem cell therapy research is addressed. A personal reflection is undertaken on how overenthusiastic translation of early experimental findings, mechanistic misconceptions, underpowered trials, and also exacerbated reliance on common binary interpretation of trial results, based on arbitrary thresholds of statistical significance, may all have contributed in some way to drive the field towards apparent stagnation, and how current and upcoming lines of investigation will likely change the face of regenerative medicine in the near future