Daria Nurzynska

Cardiac Stem Cells Possess Growth Factor-Receptor Systems That After Activation Regenerate the Infarcted Myocardium, Improving Ventricular Function and Long-Term Survival

Cardiac stem cells and early committed cells (CSCs-ECCs) express c-Met and insulin-like growth factor-1 (IGF-1) receptors and synthesize and secrete the corresponding ligands, hepatocyte growth factor (HGF) and IGF-1. HGF mobilizes CSCs-ECCs and IGF-1 promotes their survival and proliferation. Therefore, HGF and IGF-1 were injected in the hearts of infarcted mice to favor, respectively, the translocation of CSCs-ECCs from the surrounding myocardium to the dead tissue and the viability and growth of these cells within the damaged area. To facilitate migration and homing of CSCs-ECCs to the infarct, a growth factor gradient was introduced between the site of storage of primitive cells in the atria and the region bordering the infarct. The newly-formed myocardium contained arterioles, capillaries, and functionally competent myocytes that with time increased in size, improving ventricular performance at healing and long thereafter. The volume of regenerated myocytes was 2200 &mgr;m3 at 16 days after treatment and reached 5100 &mgr;m3 at 4 months. In this interval, nearly 20% of myocytes reached the adult phenotype, varying in size from 10 000 to 20 000 &mgr;m3. Moreover, there were 43±13 arterioles and 155±48 capillaries/mm2 myocardium at 16 days, and 31±6 arterioles and 390±56 capillaries at 4 months. Myocardial regeneration induced increased survival and rescued animals with infarcts that were up to 86% of the ventricle, which are commonly fatal. In conclusion, the heart has an endogenous reserve of CSCs-ECCs that can be activated to reconstitute dead myocardium and recover cardiac function.

Bone Marrow Cells Differentiate in Cardiac Cell Lineages After Infarction Independently of Cell Fusion

Recent studies in mice have challenged the ability of bone marrow cells (BMCs) to differentiate into myocytes and coronary vessels. The claim has also been made that BMCs acquire a cell phenotype different from the blood lineages only by fusing with resident cells. Technical problems exist in the induction of myocardial infarction and the successful injection of BMCs in the mouse heart. Similarly, the accurate analysis of the cell populations implicated in the regeneration of the dead tissue is complex and these factors together may account for the negative findings. In this study, we have implemented a simple protocol that can easily be reproduced and have reevaluated whether injection of BMCs restores the infarcted myocardium in mice and whether cell fusion is involved in tissue reconstitution. For this purpose, c-kit–positive BMCs were obtained from male transgenic mice expressing enhanced green fluorescence protein (EGFP). EGFP and the Y-chromosome were used as markers of the progeny of the transplanted cells in the recipient heart. By this approach, we have demonstrated that BMCs, when properly administrated in the infarcted heart, efficiently differentiate into myocytes and coronary vessels with no detectable differentiation into hemopoietic lineages. However, BMCs have no apparent paracrine effect on the growth behavior of the surviving myocardium. Within the infarct, in 10 days, nearly 4.5 million biochemically and morphologically differentiated myocytes together with coronary arterioles and capillary structures were generated independently of cell fusion. In conclusion, BMCs adopt the cardiac cell lineages and have an important therapeutic impact on ischemic heart failure.

Cardiac Stem Cell and Myocyte Aging, Heart Failure, and Insulin-Like Growth Factor-1 Overexpression

Abstract— To determine whether cellular aging leads to a cardiomyopathy and heart failure, markers of cellular senescence, cell death, telomerase activity, telomere integrity, and cell regeneration were measured in myocytes of aging wild-type mice (WT). These parameters were similarly studied in insulin-like growth factor-1 (IGF-1) transgenic mice (TG) because IGF-1 promotes cell growth and survival and may delay cellular aging. Importantly, the consequences of aging on cardiac stem cell (CSC) growth and senescence were evaluated. Gene products implicated in growth arrest and senescence, such as p27Kip1, p53, p16INK4a, and p19ARF, were detected in myocytes of young WT mice, and their expression increased with age. IGF-1 attenuated the levels of these proteins at all ages. Telomerase activity decreased in aging WT myocytes but increased in TG, paralleling the changes in Akt phosphorylation. Reduction in nuclear phospho-Akt and telomerase resulted in telomere shortening and uncapping in WT myocytes. Senescence and death of CSCs increased with age in WT impairing the growth and turnover of cells in the heart. DNA damage and myocyte death exceeded cell formation in old WT, leading to a decreased number of myocytes and heart failure. This did not occur in TG in which CSC-mediated myocyte regeneration compensated for the extent of cell death preventing ventricular dysfunction. IGF-1 enhanced nuclear phospho-Akt and telomerase delaying cellular aging and death. The differential response of TG mice to chronological age may result from preservation of functional CSCs undergoing myocyte commitment. In conclusion, senescence of CSCs and myocytes conditions the development of an aging myopathy.

Nuclear Targeting of Akt Enhances Kinase Activity and Survival of Cardiomyocytes

Abstract— Heart failure is associated with death of cardiomyocytes leading to loss of contractility. Previous studies using membrane-targeted Akt (myristolated-Akt), an enzyme involved in antiapoptotic signaling, showed inhibition of cell death and prevention of pathogenesis induced by cardiomyopathic stimuli. However, recent studies by our group have found accumulation of activated Akt in the nucleus, suggesting that biologically relevant target(s) of Akt activity may be located there. To test this hypothesis, a targeted Akt construct was created to determine the antiapoptotic action of nuclear Akt accumulation. Nuclear localization of the adenovirally encoded Akt construct was confirmed by confocal microscopy. Cardiomyocytes expressing nuclear-targeted Akt showed no evidence of morphological remodeling such as altered myofibril density or hypertrophy. Nuclear-targeted Akt significantly elevated levels of phospho-Akt and kinase activity and inhibited apoptosis as effectively as myristolated-Akt in hypoxia-induced cell death. Transgenic overexpression of nuclear-targeted Akt did not result in hypertrophic remodeling, altered cardiomyocyte DNA content or nucleation, or enhanced phosphorylation of typical cytoplasmic Akt substrates, yet transgenic hearts were protected from ischemia-reperfusion injury. Gene array analyses demonstrated changes in the transcriptional profile of Akt/nuc hearts compared with nontransgenic controls distinct from prior characterizations of Akt expression in transgenic hearts. Collectively, these experiments show that targeting of Akt to the nucleus mediates inhibition of apoptosis without hypertrophic remodeling, opening new possibilities for therapeutic applications of nuclear-targeted Akt to inhibit cell death associated with heart disease.

Activation of Cardiac Progenitor Cells Reverses the Failing Heart Senescent Phenotype and Prolongs Lifespan

Heart failure is the leading cause of death in the elderly, but whether this is the result of a primary aging myopathy dictated by depletion of the cardiac progenitor cell (CPC) pool is unknown. Similarly, whether current lifespan reflects the ineluctable genetic clock or heart failure interferes with the genetically determined fate of the organ and organism is an important question. We have identified that chronological age leads to telomeric shortening in CPCs, which by necessity generate a differentiated progeny that rapidly acquires the senescent phenotype conditioning organ aging. CPC aging is mediated by attenuation of the insulin-like growth factor-1/insulin-like growth factor-1 receptor and hepatocyte growth factor/c-Met systems, which do not counteract any longer the CPC renin–angiotensin system, resulting in cellular senescence, growth arrest, and apoptosis. However, pulse-chase 5-bromodeoxyuridine–labeling assay revealed that the senescent heart contains functionally competent CPCs that have the properties of stem cells. This subset of telomerase-competent CPCs have long telomeres and, following activation, migrate to the regions of damage, where they generate a population of young cardiomyocytes, reversing partly the aging myopathy. The senescent heart phenotype and heart failure are corrected to some extent, leading to prolongation of maximum lifespan.

CD117‐Positive Cells in Adult Human Heart Are Localized in the Subepicardium, and Their Activation Is Associated with Laminin‐1 and α6 Integrin Expression

CD117‐positive cells contributing to cardiac cell turnover in normal and pathological conditions have recently been described in adult human heart. Since the precise spatial and temporal expression of extracellular matrix proteins and their receptors is critical for organ formation, we compared the distribution of cardiac primitive CD117‐positive cells in the human adult normal and pathological hearts with ischemic cardiomyopathy, with respect to localization and expression of laminin and integrin isoforms. In the pathological hearts, CD117‐positive cells were significantly more numerous than in the normal hearts. They were localized mainly in the atria and were up to 38‐fold more numerous in the subepicardium than in the myocardium. Compared with normal hearts, most CD117‐positive cells in the subepicardium of pathological hearts were α6 integrin‐positive. Laminin‐1, typical of developing heart, was found predominantly in the subepicardium of adult heart. Immunoblotting revealed its highest expression in the normal atrium and pathological left ventricle. Both laminin isoforms reduced apoptosis and increased proliferation and migration of CD117‐positive cells in vitro with respect to control, but the effects of laminin‐1 significantly outweighed those of laminin‐2. Signaling mediated by α6 integrin was implicated in the migration and protection from apoptosis, as documented by transfection with specific small interfering RNA. These data reveal that the increase in the number of cardiac CD117‐positive cells and the expression of laminin‐1 are observed in ischemic cardiomyopathy. Subepicardial localization of CD117‐positive cells and expression of laminin‐1 and α6 integrin subunits may all correspond to the activation of regeneration involving an epithelial‐mesenchymal transition recently described in adult heart.

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.

Polyurethane-based scaffolds for myocardial tissue engineering

Bi-layered scaffolds with a 0°/90° lay-down pattern were prepared by melt-extrusion additive manufacturing (AM) using a poly(ester urethane) (PU) synthesized from poly(ε-caprolactone) diol, 1,4-butandiisocyanate and l-lysine ethyl ester dihydrochloride chain extender. Rheological analysis and differential scanning calorimetry of the starting material showed that compression moulded PU films were in the molten state at a higher temperature than 155°C. The AM processing temperature was set at 155°C after verifying the absence of PU thermal degradation phenomena by isothermal thermogravimetry analysis and rheological characterization performed at 165°C. Scaffolds highly reproduced computer-aided design geometry and showed an elastomeric-like behaviour which is promising for applications in myocardial regeneration. PU scaffolds supported the adhesion and spreading of human cardiac progenitor cells (CPCs), whereas they did not stimulate CPC proliferation after 1–14 days culture time. In the future, scaffold surface functionalization with bioactive peptides/proteins will be performed to specifically guide CPC behaviour.

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.