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Among biomaterials tested so far in cardiac tissue engineering, the decellularized native ECM (d-ECM) appears by far the most promising and appealing[1]. In order to obtain intact scaffold of d-ECM while preserving its composition, we compared the human cardiac d-ECM produced through four different protocols (named Pr1, Pr2, Pr3 and Pr4) in terms of efficiency of decellularization, composition of scaffolds and their suitability for repopulation with cardiac stem cells to develop myocardial patches. As regards efficiency of decellularization, DNA measurement revealed ineffectiveness of Pr1 and Pr3 in producing thoroughly acellular d-ECM, as they yielded scaffolds containing 46,33+5,3 and 96,7+12,2 ng of DNA/mg of tissue, respectively, while DNA content was as low as 9+2,1 in d-ECMs produced with Pr2, which includes DNase treatment, and 17,33+2.22 in the ones obtained with Pr4. Hematoxylin and Eosin staining confirmed the effectiveness of the decellularization procedure for Pr2 and Pr4, while Masson's staining revealed that Pr4 produced a d-ECM with more preserved architecture. Sirius Red, PAS and Gomori stainings documented the retention of collagen, non-collagenous proteins and elastic fibers in d-ECM from all protocols. Immunodetection of fibronectin, tenascin and laminin confirmed the retention of such ECM proteins in d-ECM, while assay for insoluble collagen and sulphated glycosaminoglycan confirmed the presence of collagen and GAGs in all protocols. Mostly due to ineffective decellularization and, as such, to mild treatment, ECM obtained through Pr3 retained more collagen and GAGs. Nevertheless, collagen content was only 1,5-fold higher than in d-ECM obtained with Pr4, which retained the highest amount of GAGs as well. Notably, GAGs protect growth factors against proteolysis, and protein array to investigate the retention of growth factors by d-ECM proved the highest efficiency of Pr4 in retaining growth factors as HGF, VEGF, TGF and IGF-1 in d-ECM. Finally, to assess suitability of d-ECM scaffolds for cell repopulation, we cultured cardiac stem cells (CSCs) on d-ECM to evaluate their viability and differentiation trend. Among all, Pr2 and Pr4 better supported cellular viability, but Pr4 provided the best environment to promote terminal differentiation towards myocardium, endothelium and smooth muscle cell lineages. Interestingly, we previously reported the inability of human cardiac stem cells to acquire fully differentiated state in vitro[2], but d-ECM supports stem cell terminal differentiation prompting progenitor cells to further proceed in differentiation towards precursor and mature phenotype. In conclusion, the composition and architecture of scaffolds of human cardiac d-ECM depend on decellularization protocol. We report here an adaptation of previously described protocol which yields highly preserved d-ECM, in terms of composition and architectures, that might be successfully be employed in regenerative medicine as biological scaffold.

Induced Pluripotent Stem cells (iPSC) are pluripotent stem cells reprogrammed from adult somatic cells. Although iPSC hold great potential for applications in regenerative medicine, technical problems, mostly related to the low efficiency of reprogramming, are yet to be solved. Since the most used cells for iPSC reprogramming are skin fibroblasts (FB), and since FB preserve positional memory, we hypothesize that the anatomic origin of FB might influence iPSC reprogramming.We isolated FB from skin of five different sites (neck, arm, thigh, breast, abdomen) of 13 patients undergoing plastic surgery or from heart wall or ascending aorta wall of the explanted heart of 3 patients receiving heart transplantation. FB from different anatomic sites and control FB from neonatal foreskin, were cultured for one week to evaluate morphology, proliferation rate and proneness to apoptosis. Additionally, expression of vimentin, cadherin, smooth muscle actin and Factor VIII was investigated to exclude the presence of other cell types. Transcriptome analysis including genes involved in stemness maintenance, embryogenesis, cell growth, activation and development, was performed by real-time PCR. Despite the similar morphology of FB from different sites, and immunopositivity for vimentin, along with the absence of other cell type markers, FB isolated from abdomen and heart had 1.5-fold higher doubling time, while FB from heart, abdomen and breast were less susceptible to apoptosis. Intriguingly, Real-Time PCR revealed that in abdomen, breast, neck, arm and heart FB genes involved in cell growth, development, proliferation, and migration, as TM4SF1, GPC4, CSPG2, DDIT4, ID1 were up-regulated, while genes regulating embryogenesis and tissue morphogenesis, like VCAN, FN1, HOXA5, CD49a were up-regulated in FB isolated from abdomen, arm and heart. However, all FBs had transcripts of markers of Mesenchymal Stem Cells (MSC), as CD105 and CD90. Our results provide evidence that human adult FB from different sites have different genetic program. Therefore, FB may respond to reprogram technology in different manner, thus affecting reprogramming efficiency. While offering novel perspective of the reprogramming technology, our study also demonstrates that abdomen and breast FB share cardiac genetic signature of cardiac FB while expressing markers of MSC and they might represent the ideal cell for cardiac reprogramming., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

The ascending aorta and pulmonary trunk develop from the common truncus arteriosus that later becomes symmetrically divided by the aorticopulmonary septum. Normally, the systolic pressure value and the diastolic pressure gradient in the aorta is much higher than that in the pulmonary artery. In the Ross procedure, patient’s diseased aortic valve is replaced with their own pulmonary valve and as a consequence the pulmonary trunk is placed in the aortic root position. Typically, a reinforcement of transposed pulmonary trunk is necessary to avoid vessel dilation. In order to investigate how the blood flow characteristics determine the vessel wall structure we examined by immunochemistry the wall of normal aorta (NA), normal pulmonary trunk (NPT), transposed pulmonary trunk (trPT) and transposed pulmonary trunk with reinforcement (trPT-R). Throughout the tunica media of NA, elastic fibers form numerous, conspicuous and orderly arranged wavy lamellae that parallel thin layers of smooth muscle cells between the internal and external elastic membrane. In the NPT, smooth muscle cells run amid collagen fibers and form layers that are thicker and irregular, with elastic fibers arranged accordingly. In the trPT, intima denudation and media disruptions were observed. In the tunica media, smooth muscle cells were abundant, but muscle fibers, with irregular profiles and no discernible alignment, were widely spaced with intervening collagen fibers. In the trPA-R, the endothelial lining was preserved and elastic fibers formed a thick and highly organized layer of concentric lamellae in the middle third of tunica media. The structure of normal aorta and pulmonary trunk, both elastic arteries, with common embryological origin, differs significantly in terms of smooth muscle and elastic sheets number and organization. The animal model of Ross procedure with pulmonary trunk in aorta position further underscores the role of blood pressure and mechanical stress in vessel wall modification., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Angiotensin II (AngII) levels are often increased in patients with cardiovascular disease and different AngII receptor (ATR) blockers are commonly used in heart failure therapy. An increase in number of cardiac primitive cells (CPCs) has been found in adult human pathological heart and these cells express AT1 and AT2 receptors (ATR). Hence, it is conceivable that AngII and ATR blockers influence CPC properties. In this study we investigated the effects of AngII and AT1R, AT2R blocking on human CPCs in vitro. CPCs were isolated from adult human hearts with ischemic cardiomyopathy (n=11) by tissue digestion and adherent CD117(+) cells immunomagnetic separation. Cells were cultured in the presence of AngII 0,1 and 1μM for 24 hr with and without pretreatment with 1μM valsartan - AT1R blocker (AT1RB), 1μM PD 123319 - AT2R blocker (AT2RB), 10μM telmisartan - AT1RB with PPAR activity, 1μM GW9662 - PPARγ blocker (PPARB). The expression of PPARγ was evaluated by electrophoresis and immunoblotting, while the proliferation of cells was evaluated by MTT assay. Administration of AngII stimulated proliferation of CPCs. The expression of PPAR γ increased 8,5 and 11,8-fold (p, Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

The Ross procedure has emerged as a popular choice for aortic valve replacement in infants and children. However, pulmonary artery (PA) autograft dilation remains the major concern; hence, several modifications of the valve implantation techniques, such as reinforcing the autografts with a tubular synthetic mesh, have been reported. With the aim to prevent dilation and permit the normal growth of the neo-aortic root following pulmonary autograft implantation, we assessed the biological effect and long term performance of an external bioresorbable reinforcement for PA autograft in an experimental Ross model in growing animals. An experimental model of translocation of the pulmonary trunk as autograft in aortic position, funded on the Hook’s law and Laplace equilibrium, has been developed and performed under cardiopulmonary bypass in young lambs. The PA without reinforcement (n=5) was compared to PA reinforced with new scaffold polymer with an external armour of Polytetrafluoroethylene. The PA autograft diameter was measured using transoesophageal echography at day 0 and at 6 months and compared to the distal aortic diameter. Pathological analysis of the PA autograft reinforced was performed at 6 months and the results were compared to those of a control group with no reinforcement (n=5) Animal weight was 25+5 kg at day 0 and 58+10 kg at 6 months and the reference aortic diameter increased from 14+1mm at day 0 to 17+2mm at 6 months. With no reinforcement, an instantaneous PA graft distension (27,4+2mm) was noted followed by an aneurysmal formation at 6 months (38+3mm). Reinforcement with scaffold polymer on polidioxanone allowed maintaining the PA graft diameter close to the reference value (17+2mm at day 0). Immunohistochemistry revealed MMP-9 overexpression indicating the induction of a matrix remodeling process that are not detectable in the control group. Mallory staining revealed elastin deposition in the reinforced PA in comparison to the collagen present in the non-reinforced group, reliably suggesting a shift towards an elastic remodeling and arterialization. PicroSirius red staining reveled in the control group collagen fibers non- homogeneously distributed with a increased cellularity indicating inflammatory infiltrates. The reinforced PA displays more organized and dense collagen fibers in the “elastic zone” of the vessel and less pronounced cellular infiltrate. In conclusion, bioresorbable external polydioxanone-based reinforcement allowed a structural rearrangement of PA autograft consisting of media reorganization with an increase in the elastic wall component. Such histological outcome arguably prevented autograft dilation and conferred enhanced mechanical properties on the PA wall, Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Chronic ischemic heart disease is characterized by an imbalance between cardiac cell death and regeneration. The local non-invasive activation of cardiac stem cells offers the possibility to influence the course of cardiomyopathy and to inhibit or reverse the pathological remodelling. In patients with coronary artery disease, low energy shock wave (SW) therapy was found to improve symptoms and ameliorate ischemia. The aim of our study was to evaluate the effects of SW on cardiac resident primitive cells in vitro. CD117(+) cells were isolated from the atria of adult human normal (n=7, males, mean age 51±5.6 years) and pathological hearts with ischemic cardiomyopathy (n=11, males, mean age 55±3.2 years), and treated with 800 shots of SW at the energy flux density of 0.05mJ/mm². Phospho-MAPK protein expression was assayed with proteome profiler array after 1 hour and total mRNA was examined by signal transduction specific PCR-based array 72 hours after the treatment. Gene expression profiling revealed in CD117(+) cells from pathological heart the down-regulation of genes relevant to the hedgehog pathway (EN1, BMP4, FOXA2), that is active in the early stages of the developmental process, or belonging to the induction and positive regulation of programmed cell death functional groups (CDKN2A, CDKN2B, CDKN1A, TP53I3, TNF), while the transcriptionally up-regulated genes included CSF2, CCL20, BIRC3, IL8, and IL1A that are involved in NFκB survival pathway and CEBPB, HK2, LEP involved in insulin pathway and myogenesis. Although SW treatment resulted in the significant increase in phospho-ERK1/2 in both cell populations, the phosphorylation of its downstream substrate RSK1 increased only in the normal cells. Similarly, in the normal cells the phosphorylation of p38-MAPKα, β and γ increased 4, 4-, 1, 2- and 4,2-fold, respectively, while only 1,3- and 2,2-fold increase in phosphorylated p38α and γ was observed in pathological cells. Moreover, the prosurvival kinase Akt1 resulted activated only in the normal cells. We conclude that the application of SW activates survival pathways and protects from apoptosis, although different signal transduction pathways can be responsible for these effects in cardiac primitive cells from normal and pathological heart with ischemic cardiomyopathy.

Cardiac Stem Cells (CSCc) contributing to cell turnover have been recently described in adult heart. The expression of extracellular matrix (ECM) proteins changes in qualitative and quantitative manner during heart remodeling. However, the influence of these changes on CSCs biology awaits investigation. The scope of the study was to evaluate the influence of fibronectin, laminin-1 and -2, collagen IV and of the natural ECM (cardiogel), obtained by culturing cardiac fibroblasts (CFs) isolated from normal (n=7) and pathological (n=8) hearts, on the biology of normal CSCs. Proliferation of CSCs, evaluated by BrdU incorporation, was 6,3-fold higher in the presence of laminin-1 and only 2-fold higher on laminin-2 with respect to control albumin. Apoptosis, detected in TUNEL assay, was as low as 1,8±0,4% on laminin-1 and was 2,8-fold, 5,4-fold and 8,5-fold higher on laminin-2, fibronectin and collagen IV, respectively. As revealed by immunofluorescence and immunoblotting, normal CFs synthesized mainly laminin-2, while pathological CFs produced more laminin-1, fibronectin, collagen IV and tenascin C. Interestingly, proliferation rate of CSCs on pathological cardiogel was 5,7-fold higher than that in the presence of normal cardiogel. In the similar manner, components of ECM from pathological heart diminished apoptosis rate with respect to cardiogel from normal heart. To assess the role of ECM on CSCs biology, we investigated the expression of and the signaling mediated by integrins. Noticeably, CSCs expressed mainly alpha-6 and alpha-2 integrin when cultured on pathological cardiogel and beta-3 integrin on normal cardiogel and, when interfering with integrin-mediated signals by specific function blocking antibodies, alpha-2 and beta-3 integrin resulted implicated in the stimulation of CSCs proliferation, while alpha-6 and alpha-2 in the protection of CSCs from apoptosis. We conclude that the components of ECM, responding to the physiological changes during heart remodeling, have profound effects on CSCs. These results should be taken into consideration when planning stem cells transplantation in the diseased hearts, since the microenvironment may determine the fate of the injected cells.

Recently, the presence of numerous cardiac stem cells in the subepicardium of the adult human heart has been described. Hence, we assessed the hypothesis that epithelial-mesenchymal transition (EMT) takes place in the adult human heart and investigated a possible link between epicardium, epicardially-derive cells (EPDCs) and cardiac stem cells. Fragments of adult human normal (n = 11) and pathological hearts with ischemic cardiomyopathy (n= 20) were examined by immunohistochemistry. In the normal hearts, cells positive for epithelial markers, cytokeratin-5/6, β-catenin, Bves, were confined to epicardium. In contrast, rare positive cells were distributed throughout the subepicardium in the pathological hearts. Noticeably, cells were absent from epicardium of diseased hearts. RT-PCR analysis of mRNA revealed that the expression of E-cadherin, cytokeratin 5, and BVES was significantly higher in the normal hearts, while that of mesenchymal markers, M-cadherin, Tie-2, and vimentin was predominant in the hearts with ischemic cardiomyopathy. Toluidine blue staining excluded that EPDCs can be CD117(+) mast cells. Next, we cultured the fragments of epicardium from the normal hearts on the specific substrate formed by extracellular matrix produced by cardiac fibroblasts in vitro, thus obtaining the outgrowth of cells with preserved epithelial phenotype. TGFβ induced cellular and molecular changes typical of epithelial-mesenchymal transition, which were confirmed by morphological study, immunofluorescence and RT-PCR. In particular, the expression of E-cadherin and cytokeratin-5 in EPDCs diminished 2.27-fold and 5-fold, respectively. Moreover, mesenchymal gene RT-PCR products of vimentin and M-cadherin were readily detected, with the 1.84-fold increase of the latter. Interestingly, 32±1.8%; of EPDCs were CD117(+). These cells expressed markers of cardiomyocyte, endothelial and smooth muscle cell cardiac progenitors and precursors.In conclusion, our results provide evidence that cardiac stem cells originate from epicardial cells which undergo EMT in the adult human heart. EPDCs can be promising candidates for clinical application in ischemic hearts – the more so considering that a subset of them represents cardiac CD117(+) stem cells.

According to recent hypothesis, pathological processes may deplete the heart of stem cells and uncertainty still prevails as to whether functionally competent primitive cells can be obtained also from failing hearts. The scope of the present study was to assess proliferation, apoptosis, differentiation and commitment of cardiac primitive cells resident in normal (n=8) adult human hearts and in hearts with ischemic cardiomyopathy (n=8). Immunofluorescence and immunoblotting of CD117(+) cells isolated from normal and pathological hearts revealed higher expression of cardiac-specific nuclear and cytoplasmic markers in the latter. Proliferation of CD117(+) cells isolated from hearts with ischemic cardiomyopathy was 3.2±0.7-fold higher (p‹0.005), while the apoptosis induced by oxidative stress was 1.8±0.2-fold higher (p‹0.005) when compared with cells from normal hearts. Gene expression was examined by stem cells specific PCR-based microarray. We confined our analysis to genes with at least a 1.7 fold differential expression and identified 38 downregulated and 39 upregulated genes in CD117(+) cells from failing hearts. The downregulated genes included snail1 homolog, jagged-1 and ephrin b1 that are involved in the early stages of the developmental process; mdm2, p15 and p16 belonging to the cell cycle control functional group, as well as genes from the neurogenesis, skeletal development, bone remodeling and cartilage development functional classes. Upregulated genes, among which OTF2, endothelin receptor, CD105 and MRG1, were involved in developmental maturation, mesenchymal cell differentiation, heart development and circulatory system processes. One major finding emerging from the analysis is the acquired capability of cells from pathological heart of responding to wounding, as well as the upregulation of the response to external stimuli and stress. In comparison with normal cells, those from pathological heart become readily committed to cardiac cell lineages, at the expense of stemness and multipotentiality. Moreover, the epithelial-mesenchymal transition (EMT), which has been proposed as the mechanism behind cardiac stem cells origin, resulted activated, as well as known inducers of EMT, TGFβ and HGF signaling pathways. Cardiac stem cells in the normal and in the pathological heart differ in several respects and the differences reflect the activation of cardiac stem cells pool in the chronic pathological conditions. Given the apparent failure of intrinsic heart regeneration, further studies are warranted to optimize the strategies for cardiac stem cells application in regenerative medicine

The notion of the presence of stem and progenitor cells in the adult human heart imposes that all currently used treatments, as well as those in the experimental phase of the study, should be revisited with regards to effects on these cardiac cell population. In fact, novel therapies offer the possibility of inhibition or even reversal of heart failure progression by the mobilization and activation of cardiac stem and progenitor cells, as can be the case with extracorporeal cardiac shock wave (SW) therapy, suggested for patients with myocardial ischemia. The scope of the present study was to evaluate the effects of low and high energy SW treatment on cardiac primitive cells in vitro. CD117(+) cells have been isolated from age-matched adult human normal (n=4, males, mean age 51±5.6 years) and pathological hearts with end-stage ischemic cardiomyopathy (n=4, males, mean age 55±3.2 years). Cells have been treated with 800 shots of SW at the energy flux density of 0.05mJ/mm2 and 0.1mJ/mm2. After one week of culture, cardiac cell lineages were characterized by immunofluorescence and immunoblotting, and their total mRNA was examined by stem cell-specific PCRbased microarray. Above all, the effects of high energy treatment differed profoundly from those observed at low energy, with none the same gene up- or downregulated in any of the groups studied. Moreover, cells isolated from pathological hearts were positively influenced in the more evident manner. In fact, low energy treatment of pathological cells induced the activation of a pool of transient amplifying progenitors of cardiomyocytes (expressing α-sarcomeric actin), endothelial (von Willebrand factor) and smooth muscle cells (smooth muscle actin). In these cell population, we observed the upregulation of mRNA of 14 genes involved in the cytoskeleton biogenesis/organization, mitotic spindle formation and cell motility. In contrast, high energy SW application resulted in upregulation of 5 genes, mostly involved in the extracellular matrix- and cell-cell adhesion, and downregulation of 15 genes regulating protein kinase activity, signal transduction and cell-cell signalling. As regards cells from normal heart, remarkably only high energy treatment induced activation of endothelial cell precursors. On the basis of the above data it is possible to predict that timing (between onset and end-stage of disease) and energy of the SW treatment can have profound influence on the therapy outcome in ischemic heart disease. Further pre-clinical studies of these variables are warranted before broad introduction of the method into clinical practice.

Growing number of studies indicate epicardium-derived cells (EPDCs) as cardiac stem cells. While it is beyond doubt that these cells contribute to the normal developmentof the heart during organogenesis, it remains an open question whether mesothelial epicardial cells can undergo epithelial–mesenchymal transition, giving origin to cardiac primitive cells, in the adult human heart. We examined epicardium and subepicardium of the atria of human adult normal (n=11, mean age 41±12 years, 7 males, 4 females) and pathological hearts with chronic heart failure due to ischemic cardiomyopathy (n=22, mean age 55±5.5 years, 14 males, 8 females, mean ejection fraction 25±1%). Strikingly, only the normal adult human hearts were layered with epicardial cells. On the contrary, cell nuclei were absent from the surface lining of the diseased hearts. While normal epicardium resulted positive for cytokeratin 5/6, E-cadherin and Bves, in the pathological hearts the cells with epithelial markers were distributed among loose connective tissue of the subepicardium. Remarkably, these cells were CD117-positive and, when compared with subepicardium of normal heart, their number was 8.7-fold (p‹0.005) higher in the subepicardium of hearts with ischemic cardiomyopathy. In the PCR-based array of CD117-positive cells isolated from normal and pathological hearts, TGFβ and HGF receptor signaling pathways (both known inducers of EMT) resulted activated in the latter. Considering the hypothesis that EPDCs contribute to the cardiac CD117-positive cells population in the adult heart, fragments of epicardium of adult human cardiac atria were cultured on the extracellular matrix produced by cardiac fibroblasts, to obtain the outgrowth of the mesothelial cells forming epithelial sheets. When stimulated with TGFβ and HGF, intercellular contacts were lost and cells acquired mesenchymal characteristics and CD117 expression. Altogether, these results indicate that EPDCs enrich the pool of cardiac primitive cells that contribute to the regenerative properties of the adult human heart. It could be reasonably argued that CD117-positive and EPDCs represent the same primitive cell population.

Doxorubicin is one of the most active anti-cancer drugs, widely used for the treatment of several malignancies, however its clinical implementation is limited by high incidence of cardiovascular events, most serious of them being the cardiomyopathy followed by congestive heart failure. Known studies of the mechanisms of doxorubicin toxicity focused on cardiomyocyte damage. But then cardiac primitive cells are considered to provide the adult heart with a substantial growth reserve determining the function of the heart throughout life. The role of this cell population in the myocardial response to different pathologic stimuli has been documented in animals and humans, suggesting cardiac primitive cells as possible pathophysiologic target in cardiac diseases. We advance the hypothesis that cardiotoxicity of doxorubicin may be due to its effects on cardiac primitive cells. Hence, we examined doxorubicin toxicity on this cell population. CD117-positive cells isolated from adult human normal atria were incubated with increasing concentrations of doxorubicin hydrochloride (0.1, 0.5 and 1μM), followed by the evaluation of proliferation and apoptosis after 12, 24, 48 and 60 hours. Proliferation was evaluated by BrdU incorporation and its rate dropped from 12.84±1.83% (control, n=4) to 2.41±0.27% (24 hours, 1μM, n=4, p

The cumulative dose-dependent toxicity of doxorubicin, with the late development of the chronic heart failure that does not respond to digitalis, imposes serious limits on cancer treatment and often leads to heart transplantation in relatively young patients. Known studies of the mechanisms of doxorubicin toxicity focused on cardiomyocytes damage. According to the new notion of cardiac tissue biology, cardiac primitive cells are considered to provide the adult heart with a substantial growth reserve determining the function of the heart throughout life. Hence, numerous factors that can influence the subtle balance between cardiac primitive cells death and survival, can be implicated in the development and progression of cardiomyopathy and heart failure. The scope of the study was to examine the effects of doxorubicin on human adult CD117-positive cardiac primitive cells in vitro. Moreover, the role of dexrazoxane, a chelating agent with cardioprotective action, was revisited with respect to the reduction of doxorubicin toxicity on cardiac primitive cells. CD117-positive cells were incubated with increasing concentrations of doxorubicin hydrochloride (0,1; 0,5 and 1,0μM), followed by the evaluation of proliferation and apoptosis after 12, 24 and 48 hours. Proliferation was evaluated by BrdU incorporation and it decreased at all time-points, with the most significant effects after 24 hours, when its rate dropped from 12,84±1,83% (n=4) to 2,41±0,27% (n=4, p

CD117(+) cells contributing to the cardiac cell turnover have been recently described in the adult heart, together with numerous factors that influence their characteristics in the normal and pathological conditions. It is known that the precise spatial and temporal expression of extracellular matrix proteins and their receptors is critical for proper organ formation during organogenesis. Hence, the cardiac regeneration cannot be accomplished without the involvement of extracellular matrix. The scope of the study was to evaluate the influence of fibronectin, laminin-1, laminin-2 and collagen IV on cardiac primitive CD117(+) cells isolated from human adult heart. Moreover, cardiac fibroblasts from human adult normal and pathological hearts with ischemic cardiomyopathy were isolated and cultured to obtain the natural extracellular matrix (cardiogel) in vitro. After non-enzymatic removal of fibroblasts, this substrate was used to compare the proliferation and apoptosis of CD117(+) cells in the presence of extracellular matrix typical of normal and pathological heart. Proliferation of CD117(+) cells, evaluated by BrdU incorporation, was as low as 4,4±0.9% (n=4) in the presence of albumin and reached maximum rate of 27,7±4,7% (n=4) on laminin-1 and minimum of 8,6±0,2% (n=3) on laminin-2. Apoptosis, detected in TUNEL assay, was minimum in the presence of laminin-1 (1,8±0,4%, n=4) when compared with laminin-2 (5,2±1,1%, n=4), fibronectin (9,8±2,4%, n=4) and collagen IV (15,4±2,6%, n=4). The components of cardiogel were characterized by immunofluorescence and immunoblotting. Cardiac fibroblasts from normal heart synthesized extracellular matrix rich in laminin-2, while those from the heart with chronic ischemic cardiomyopathy produced more laminin-1, fibronectin, collagen IV and tenascin C. Interestingly, when normal CD117(+) cells were cultured on the cardiogel from pathological heart, their proliferation rate was 5,7-fold higher (n=5, p

According to the recent findings, CD117(+) cells reside in the subepicardium and epicardially-derived cells may represent a population of cardiac primitive cells. We have previously shown the presence of laminin-1, typical of developing heart, in the subepicardium of human adult heart with chronic ischemic cardiomyopathy. Hence, the hypothesis arises that laminin-1 may influence cardiac primitive cells biology. CD117(+) cells from adult human heart were cultured on laminin-1, laminin-2 or albumin. Specific human alpha6 integrin small interfering RNA (siRNA) was used to examine the contribution of alpha6 integrin and laminin interactions to the cell proliferation, apoptosis and migration in vitro. Both laminin-1 and laminin-2 increased the proliferation of cells, as evaluated with BrdU incorporation. In the presence of laminin-2 the proliferation rate reached 7.9±0.4% (n=4), whereas during the culture of cardiac primitive cells on laminin-1 the incorporation of BrdU was over 4-fold higher (n=4, p

During heart morphogenesis a subset of epicardial cells undergoes an epithelial-mesenchymal transition and invades myocardium contributing to the formation of myofibroblasts, vascular endothelium and smooth muscle cells. Recently, the presence of resident stem cells able to give rise to the cells of cardiac lineages was observed in the adult human heart, raising questions concerning their origin and biology. The scope of the present study was to investigate whether the epithelial-mesenchymal transition takes place in the adult human heart, generating the population of cardiac stem cells, and which molecular factors are involved in its initiation and progression. Since the subepicardial space is rich in extracellular matrix components and cytokines, constituting the specific environment for epicardially derived cells, cardiac fibroblasts obtained from the subepicardium layer of adult human atrium were cultured to create the most appropriate conditions for the in vitro study. After the non-enzymatic removal of cells, the presence of extracellular matrix proteins - fibronectin, collagen III and IV, tenascin and laminin - was confirmed by immunofluorescence. This substrate was used to obtain and culture the sheets of epithelial cells from the epicardium of normal adult human atria. The epithelial phenotype of the cells was confirmed by the positive immunolabeling of E-cadherin and β-catenin at the intercellular junctions and cytokeratin in the cytoplasm. While in the presence of basic fibroblast growth factor (10 ng/ml) or platelet-derived growth factor-BB (40 ng/ml) the epithelial sheets remained intact, the addition of epidermal growth factor (40 ng/ml), hepatocyte growth factor (40 ng/ml) or transforming growth factor beta (0,5 ng/ml) resulted in the change of cell characteristics. The intercellular contacts were lost and the cells acquired spindle-like shape and vimentin expression. When induced to differentiate into endothelial, smooth muscle cells or cardiomyocytes, these cells expressed factor VIII, smooth muscle actin or myosin heavy chain α/β, respectively. The results of our study indicate that in the specific conditions epithelial-mesenchymal transition takes place in the adult human epicardial cells. We suggest that epicardially derived cells enrich the pool of cardiac primitive cells and contribute to the regenerative properties of the heart. A better understanding of complex interactions of the extracellular matrix proteins and growth factors with their specific receptors on the cell surface will be pivotal for progressing therapeutic cardiac regeneration.

Cardiac cells and extracellular matrix (ECM) are reciprocally related and their characteristics are modified in response to developmental or pathophysiological cues. Adult human cardiac tissue regeneration mediated by cardiac stem cells (CSCs) is strictly regulated and, hypothetically, impaired by the ECM-CSC signalling in the pathological conditions. To test this hypothesis, we isolated cardiac fibroblasts (CFs) and CSCs from the atria of age-matched adult human normal (n=9) and pathological hearts (ischemic cardiomyopathy, n=11). The CFs were cultured in order to obtain ECM coating and conditioned medium, which were characterized by immunoblotting and ELISA, respectively. Next, we examined the effects of CF-derived ECM and CF-conditioned medium on normal and pathological CSC proliferation, apoptosis, and migration in vitro. The ECM produced by CFs from normal heart was composed mainly of fibronectin, laminin α2 and collagen I, while that produced by CFs from hearts with ischemic cardiomyopathy contained also laminin α1 and tenascin X. Compared to the normal CF-conditioned medium, that conditioned by pathological CFs contained twice as much IGF1 and HGF, and it stimulated proliferation and migration, while reducing apoptosis of CSCs. In the presence of pathological CF-derived ECM, there was a nearly 2-fold increase (p, Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Il cuore umano adulto possiede una riserva di cellule staminali, CD117 positive, che sono capaci di differenziare nelle tre principali linee cellulari cardiache, contribuendo al turnover cellulare. Lo scopo di questo lavoro è stato quello di chiarire, in vitro, gli effetti di una ECM naturale (Cardiogel), ottenuta dalla coltura di fibroblasti isolati da cuori umani adulti normali e da cuori espiantati per insufficienza cardiaca post-ischemica, sulla biologia delle cellule primitive cardiache (CPC) CD117 positive (+) anch’esse isolate da cuori umani adulti normali e post-ischemici.