From cardiogenesis to cardiac regeneration : focus on epicardium-derived cells
Winter, E.M.
Citation
Winter, E. M. (2009, October 15). From cardiogenesis to cardiac regeneration : focus on epicardium-derived cells. Retrieved from https://hdl.handle.net/1887/14054
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Summary
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SummaryIn this thesis, the potential of adult epicardium-derived cells (EPDCs), solitary or combined with cardiomyocyte progenitor cells (CMPCs), and of mesenchymal stem cells (MSCs) has been explored with regard to cardiac repair. Additionally, various applications of magnetic resonance imaging (MRI) for stem cell research in the infarcted mouse heart have been investigated.
Chapter 1 provides a general introduction to this thesis. Embryonic heart development, cardiac stem cell therapy and methods for functional evaluation of the infarcted mouse heart are addressed.
Cardiogenesis comprises the contribution of two heart-forming fields that sequentially add to the developing heart. Promising stem and progenitor cells detected in the adult heart might be remnants of these primitive structures. The concept of totipotency, pluripotency, and multipotency are introduced, as are various adult stem cells.
Chapter 2 reviews current knowledge on EPDCs. The proepicardial organ (PEO), a specific component of the second heart forming field (SHF), gives rise to the epicardial layer of the heart. EPDCs are generated from this epicardial sheet by epithelial-mesenchymal transformation (EMT), after which they invade the myocardial wall. The EPDCs contribute to the developing heart through delivering interstitial fibroblasts, adventitial fibroblasts and coronary smooth muscle cells, thereby regulating important processes like formation of the compact myocardium, Purkinje fibers, endocardial cushions and vessels. It is unknown whether EPDCs are still generated in the adult heart, and whether they might be able to recapitulate their embryonic program in favor of the ischemic heart.
In Chapter 3 the therapeutic application of human adult EPDCs for cardiac repair has been
investigated. Transplantation of human adult EPDCs into the artificially infarcted mouse myocardium improved cardiac function when compared to control vehicle injection. Ejection fraction (EF) was increased in the EPDC-recipients at week 2 after myocardial infarction (MI), and it remained enhanced up to week 6 when the scar is definitively formed. Similarly, adverse remodeling was attenuated in case of EPDC transplantation, indicated by decreased end-systolic volume (ESV) and end-diastolic volume (EDV), and survival was significantly higher. The massively engrafted EPDCs acquired a fibroblast-like phenotype and were located in the infarcted area of the left ventricle, aligned with the surrounding cells. The EPDCs appeared to stimulate the surrounding host tissue in a paracrine manner. This was indicated by the fact that i) the reduced volume expansion was already observed at day 2 after surgery, by which time new tissue could not have been regenerated, ii) the engrafted EPDCs themselves did not form new cardiomyocytes or endothelial cells, while iii) wall thickness and vascular density (mouse origin) were increased. Moreover, DNA-damage repair of the endogenous host tissue was enhanced in the EPDC-recipients.
Chapter 4 further explored the paracrine effect of adult EPDCs on the infarcted mouse myocardium, with a functional and histological evaluation at day 2, 4 and 7 after transplantation. Injected human adult EPDCs preserved left ventricular function, as opposed to potential restoration of functional loss, mainly through early but largely undefined paracrine signaling. Cardiac healing in EPDC-recipients was characterized by a temporal shift forward of natural Wilms’ Tumor 1 (WT1) expression, a marker for undifferentiated EPDCs. WT1 was detected in the activated cubic mouse epicardium and in cells scattered throughout the infarcted area already at day 2 (control group: day 4), suggesting advanced invasion of newly formed EPDCs from the host epicardium. Viral labeling experiments further substantiated this proposition by revealing that new EPDCs are indeed generated from the native tissue in case of myocardial infarction.
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SummaryIn Chapter 5 the hypothesis that co-transplantation of human adult ‘instructive’ EPDCs and
‘constructive’ CMPCs might synergistically improve cardiac function after myocardial infarction (MI), was studied. In vitro experiments revealed a mutual influence of EPDCs and CMPCs on proliferation, migration and production of various growth factors. Combined injection of EPDCs and CMPCs indeed further improved left ventricular function at 6 weeks after MI compared to single EPDC or CMPC transplantation, which themselves each enhanced cardiac performance compared to control vehicle injection. In contrast to the hypothesis, however, the synergistic effect of EPDCs and CMPCs was not instigated by both support of the host tissue as well as cardiomyocyte formation, respectively, but by paracrine signaling on the surroundings only. The engrafted human cells did not differentiate into cardiomyocytes or endothelial cells, but wall thickness and vascular density were significantly increased when EPDCs and CMPCs were transplanted as a mixture. With total injected cell number and graft volumes being similar among groups, it was postulated that EPDCs and CMPCs acted through different but complementary paracrine mechanisms.
Chapter 6 concerns the therapeutic application of MSCs from ischemic heart disease patients.
Transplantation of MSCs, which were harvested from patients suffering from cardiovascular disease, into the infarcted mouse heart significantly increased left ventricular ejection fraction at day 14 after MI compared to control vehicle injection. This functional improvement was accompanied by a reduced extent of infarct wall thinning, as well as enhanced blood vessel density (mouse origin) in the scar area of the MSC group. The engrafted MSCs did not express stringent smooth muscle cell or cardiomyocyte markers, excluding contribution of newly generated human cardiomyocytes to the functional benefit.
Chapter 7 provides a head-to-head comparison of two different methods that enable functional assessment of the infarcted mouse heart, being MRI and pressure-volume loops by conductance catheter (CC). Differences between hearts with and without MI were reliably detected by both approaches, but left ventricular volumes and ejection fraction were consistently lower by CC than by MRI. Sensitivity and specificity were good for either technique, with superior outcome for MRI.
MRI and CC could both be considered highly valuable for evaluation of murine cardiac function, recommending MRI for longitudinal studies and when detailed anatomic information is required, and CC when advanced analysis of cardiac function is needed.
Chapter 8 addresses application of iron-labeling for in vivo stem cell tracking by MRI. Dead and living iron-loaded EPDCs were transplanted into the infarcted mouse heart of the immunodeficient NOD/
scid mouse, and imaged with a 9.4T animal MRI until week 6 after surgery. In each heart determined, iron-loaded cells could be recognized on the MRI scans as voids within the left ventricular wall. They did not change in size, localization or number over time. Histological determination revealed that the spots observed on the MRI images corresponded with iron particles present within intact cells in the heart tissue. For the dead-EPDC recipients this population of iron-containing cells consisted entirely of macrophages that had phagocytosed the iron. The group having received living EPDCs exhibited iron-loaded macrophages as well as iron-positive engrafted EPDCs. Since the MRI could not discriminate between healthy, initially successfully engrafted and dead phagocytosed iron-loaded stem cells, the method can not be considered appropriate for in vivo tracking of living stem cells. Iron- labeling is, however, valuable to evaluate whether transplantation has been successful, to visualize the position of injection, and to pursue this area over time.
Chapter 9 provides a general discussion on the data presented in this thesis. Using cardiac development as basis for stem cell research design, application of various mesoderm originated adult cells, i.e. MSCs, EPDCs and CMPCs, is discussed in the light of cardiac repair. Additionally, the feasibility of different methods to functionally assess the infarcted mouse heart is addressed.
