Veronica Romano

Epithelial-mesenchymal transition of epicardial mesothelium is a source of cardiac CD117-positive stem cells in adult human heart

Epithelial-mesenchymal transition is implicated in the remodelling of tissues during development and in the adult life. In the heart, it gives origin to progenitors of fibroblasts, coronary endothelium, smooth muscle cells, and cardiomyocytes. Moreover, epicardially-derived cells determine myocardial wall thickness and Purkinje fibre network. Recently, the presence of numerous cardiac stem cells in the subepicardium of the adult human heart has been described and the hypothesis that epicardially-derived cells can contribute to the population of cardiac stem cells in the adult heart has been advanced. In an effort to test this hypothesis and establish a possible link between epicardium, epicardially-derived cells and cardiac stem cells in the adult human heart we have examined epicardial mesothelial cells in the normal and pathological adult human heart with ischemic cardiomyopathy in vivo and we have induced and documented their epithelial-mesenchymal transition in vitro. Noticeably, epicardial cells were missing from the surface of pathological hearts and the cells with the expression of epithelial and mesenchymal markers populated thick subepicardial space. When the fragments of epicardium from the normal hearts were cultured on the specific substrate formed by extracellular matrix derived from cardiac fibroblasts, we obtained the outgrowth of the epithelial sheet with the mRNA and protein expression characteristic of epicardium. TGFβ induced cellular and molecular changes typical of epithelial-mesenchymal transition. Moreover, the epicardially-derived cells expressed CD117 antigen. Thus, this study provides evidence that cardiac stem cells can originate from epithelial-mesenchymal transition of the epicardial cells in the adult human heart.

Epicardial cells are missing from the surface of hearts with ischemic cardiomyopathy: A useful clue about the self-renewal potential of the adult human heart?

The search for ideal cell candidate for heart regeneration, as well as for putative cardiac stem cell responsible for cardiac tissue homeostasis, is occupying both basic scientists and clinicians. Growing number of studies and publications indicate epicardium-derived cells as cardiac stem cells. While it is beyond doubt that these cells contribute to normal development of the heart during organogenesis, it remains an open question whether mesothelial epicardial cells can preserve their embryonic potential and if they can undergo epithelial-mesenchymal transition, giving origin to cardiac cell lineages, also in the adult human heart. Recent observations in vitro confirm this hypothesis, but direct evidence from the adult human heart is difficult to obtain. We report the absence of epicardial cells from the surface of adult human hearts with ischemic cardiomyopathy and the accumulation of cells with epithelial and mesenchymal markers in the subepicardium. We argue that these findings may correspond to the activation of the epithelial-mesenchymal transition in the chronic pathological conditions requiring cardiac cell regeneration, followed by epicardial cell pool exhaustion. Hence, observation of the epicardium of patients with cardiovascular disease, although not offering immediate diagnostic advantage, could provide some urging answers concerning the self-renewal potential of the adult heart.

Flatfoot in children: anatomy of decision making

Concern about a childs foot posture is a common reason for frequent consultations for an array of health care professionals; sports medicine specialists are often the first to recognize and advise on foot pathology. In the decision making process, it is essential to distinguish between the different types of flatfoot deformity: paediatric or adult, congenital or acquired, flexible or rigid. Although flatfoot in children is a common finding, evidence for the techniques of the reliable and reproducible assessment of the foot posture is scant. This general review presents the factors involved in the forming and supporting of the foot arches, discusses the protocols useful in the evaluation of the foot posture, and indicates how to differentiate between flatfoot cases needing treatment and cases that need only reassurance.

Cardiac shock wave therapy: assessment of safety and new insights into mechanisms of tissue regeneration

Although low‐energy extracorporeal cardiac shock wave (ECSW) therapy represents an attractive non‐invasive treatment option for ischaemic heart disease, the precise mechanisms of its action and influence on the cardiac tissue remain obscure. The goal of this study was to evaluate the effects of SW application on cardiac function and structure. Four‐month‐old Fisher 344 rats were subjected to ECSW therapy. Echocardiographic measurements of cardiac function were performed at baseline and at 1 and 3 months after treatment. Signs of inflammation, apoptosis and fibrosis were evaluated by immunohistochemistry in the control and treated hearts. ECSW application did not provoke arrhythmia or increase the troponin‐I level. At all time points, the left ventricular ejection fraction and fractional shortening remained stable. Histological analysis revealed neither differences in the extracellular matrix collagen content nor the presence of fibrosis; similarly, there were no signs of inflammation. Moreover, a population of cardiac cells that responded eagerly to ECSW application in the adult heart was identified; c‐kit–positive, Ki67‐positive, orthochromatic cells, corresponding to cardiac primitive cells, were 2.65‐fold more numerous in the treated myocardium. In conclusion, non‐invasive ECSW therapy is a safe and effective way of activating cardiac stem cells and myocardial regeneration. Because many factors influence cellular turnover in the ischaemic myocardium during the course of ischaemic heart disease, cardiac remodelling, and heart failure progression, studies to identify the optimal treatment time are warranted.

Cardiac Fibroblast-Derived Extracellular Matrix (Biomatrix) as a Model for the Studies of Cardiac Primitive Cell Biological Properties in Normal and Pathological Adult Human Heart

Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM) change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease) and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix), composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

Cardiac primitive cells become committed to a cardiac fate in adult human heart with chronic ischemic disease but fail to acquire mature phenotype: genetic and phenotypic study

Adult human heart hosts a population of cardiac primitive CD117-positive cells (CPCs), which are responsible for physiological tissue homeostasis and regeneration. While the bona fide stem cells express telomerase, their progenies are no longer able to preserve telomeric DNA; hence the balance between their proliferation and differentiation has to be tightly controlled in order to prevent cellular senescence and apoptosis of CPCs before their maturation can be accomplished. We have examined at cellular and molecular level the proliferation, apoptosis and commitment of CPCs isolated from normal (CPC-N) and age-matched pathological adult human hearts (CPC-P) with ischemic heart disease. In the CPC-P, genes related to early stages of developmental processes, nervous system development and neurogenesis, skeletal development, bone and cartilage development were downregulated, while those involved in mesenchymal cell differentiation and heart development were upregulated, together with the transcriptional activation of TGFβ/BMP signaling pathway. In the pathological heart, asymmetric division was the prevalent type of cardiac stem cell division. The population of CPC-P consisted mainly of progenitors of cardiac cell lineages and less precursors; these cells proliferated more, but were also more susceptible to apoptosis with respect to CPC-N. These results indicate that CPCs fail to reach terminal differentiation and functional competence in pathological conditions. Adverse effects of underlying pathology, which disrupts cardiac tissue structure and composition, and cellular senescence, resulting from cardiac stem cell activation in telomere dysfunctional environment, can be responsible for such outcome.

Localization and origin of cardiac CD117-positive cells: identification of a population of epicardially-derived cells in adult human heart

During heart morphogenesis, epicardial cells undergo epithelial-mesenchymal transition giving origin to a population of epicardially derived cells that play a crucial role in the development of most cardiac cell lineages. Considering the hypothesis that epithelial-mesenchymal transition of epicardial mesothelium can generate cardiac primitive cells in the adult heart, we have examined in vivo and in vitro the epicardium and subepicardium of normal human adult hearts and of pathological hearts from patients with chronic ischemic heart failure for the presence of CD117-positive cells with epithelial and mesenchymal markers expression. The number of CD117-positive cells increased significantly in the subepicardium of pathological hearts and sloped down towards myocardium, remaining still elevated with respect to normal hearts. While cells with typical epithelial proteins expression formed an intact layer on the surface of the normal hearts, CD117-positive cells were localized mainly in the subepicardium and expressed mesenchymal markers in the pathological hearts. Epithelial-mesenchymal transition, induced in vitro by several growth factors known to accumulate in the ischemic myocardium, gave origin to epicardially-derived cells with CD117 expression. These data support the hypothesis of epicardial origin of cardiac primitive cells in the adult human heart.

Analysis of SOD3 and Akt in ascending aortic aneurysm

Ascending aortic aneurysm (AsAA) is divided into three different forms: syndromic, familial non-syndromic, and degenerative. Bicuspid aortic valve (BAV), occuring in 2% of the population, is the most frequent cardiac congenital abnormality, associated to AsAA. All the different forms of AsAAs are a consequence of cystic medial necrosis (CMN), characterized by apoptotic loss of smooth muscle cells (SMCs), fragmentation of elastic and collagen fibers and increased accumulation of mucoid material. The extracellular superoxide dismutase (SOD3) is a Cu/Zn enzyme, affecting redox state and homeostasis of extracellular matrix (ECM) (1). Moreover, the outsidein signalling from ECM modulates intracellular pathways regulating many cellular functions. The multifunctional Akt pathway affects survival and cellular proliferation and has important effects on the cardiovascular function. In this study we examined the relevance of SOD3 and Akt in AsAA pathogenesis. To this aim, the SOD3 and Akt protein levels were evaluated in normal ascending aortic tissues (n=6) and in tissues from AsAAs associated both to tricuspid aortic valve (TAV) (n=6) and BAV (n=6); moreover, we measured SOD3 activity in sera from healthy donors and patients with AsAA. Our data showed a reduction of SOD3 and phospho-Akt (pAkt) protein levels in AsAAs from BAV patients compared to normal donors; on the other hand, no differences emerged in SOD3 activity. Furthermore, immunohistochemical analysis performed on normal and pathological ascending aortic tissues showed a SOD3 immunostaining in both extracellular space and tunica media cells from normal ascending aortic tissues; conversely, no SOD3 immunostaining was detected in AsAAs tissues from both TAV and BAV patients. Our data show that SOD3 and pAkt could be associated to AsAA pathogenesis and suggest a link between ECM homeostasis and Akt survival pathway.

Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts

Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed.

Optimization Of Human Heart Decellularization Method For Cardiac Regenerative Medicine

Extracellular matrix (ECM) is an intricate mesh of collagenous and non-collagenous proteins, whose presence and amount vary according to type of tissue. ECM drew the attention of regenerative medicine scientists as natural scaffold suitable for stem cell delivery into damaged tissues. Although a multitude of protocols and combinations of chemical agents and physical methods have been tested and proved effective in the decellularization of human heart, none of the ones tried in our setting fulfilled the goal of obtaining a structurally preserved cardiac decellularized ECM (d-ECM). While testing already described procedures, we made several adjustments that led to the development of a novel, simpler and robust protocol to decellularize adult human heart. Specifically, we decellularized cardiac samples of the free wall of both ventricles of adult human hearts scaled down to fit into embedding cassettes used to avoid stirring stress and preserve structure. To shorten the procedure, a combination of SDS, Triton X-100 and antibiotics was used in simple and fast two-step protocol. After decellularization, d-ECM was fixed and processed for histological study or snap-frozen for molecular biology analysis or cytocompatibility test in vitro. Histochemistry and immunoistochemistry confirmed the absence of nuclei and the preservation of architecture and composition of d-ECM. Further, while DNA content in d-ECM was well below accepted standards, sGAG, elastin and growth factors were retained and d-ECM scaffolds supported cardiac primitive cell engraftment and survival in vitro. Hence, according to our evidence, our protocol is simple, fast, effective and is worth improving for clinical translation.