Antonella De Angelis

Human cardiac stem cells

The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.

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.

Diabetes Promotes Cardiac Stem Cell Aging and Heart Failure, Which Are Prevented by Deletion of the p66shc Gene

Diabetes leads to a decompensated myopathy, but the etiology of the cardiac disease is poorly understood. Oxidative stress is enhanced with diabetes and oxygen toxicity may alter cardiac progenitor cell (CPC) function resulting in defects in CPC growth and myocyte formation, which may favor premature myocardial aging and heart failure. We report that in a model of insulin-dependent diabetes mellitus, the generation of reactive oxygen species (ROS) leads to telomeric shortening, expression of the senescent associated proteins p53 and p16INK4a, and apoptosis of CPCs, impairing the growth reserve of the heart. However, ablation of the p66shc gene prevents these negative adaptations of the CPC compartment, interfering with the acquisition of the heart senescent phenotype and the development of heart failure with diabetes. ROS elicit 3 cellular reactions: low levels activate cell growth, intermediate quantities trigger cell apoptosis, and high amounts initiate cell necrosis. CPC replication predominates in diabetic p66shc−/−, whereas CPC apoptosis and myocyte apoptosis and necrosis prevail in diabetic wild type. Expansion of CPCs and developing myocytes preserves cardiac function in diabetic p66shc−/−, suggesting that intact CPCs can effectively counteract the impact of uncontrolled diabetes on the heart. The recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell disease in which abnormalities in CPCs define the life and death of the heart. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other.

Local Activation or Implantation of Cardiac Progenitor Cells Rescues Scarred Infarcted Myocardium Improving Cardiac Function

Ischemic heart disease is characterized chronically by a healed infarct, foci of myocardial scarring, cavitary dilation, and impaired ventricular performance. These alterations can only be reversed by replacement of scarred tissue with functionally competent myocardium. We tested whether cardiac progenitor cells (CPCs) implanted in proximity of healed infarcts or resident CPCs stimulated locally by hepatocyte growth factor and insulin-like growth factor-1 invade the scarred myocardium and generate myocytes and coronary vessels improving the hemodynamics of the infarcted heart. Hepatocyte growth factor is a powerful chemoattractant of CPCs, and insulin-like growth factor-1 promotes their proliferation and survival. Injection of CPCs or growth factors led to the replacement of approximately 42% of the scar with newly formed myocardium, attenuated ventricular dilation and prevented the chronic decline in function of the infarcted heart. Cardiac repair was mediated by the ability of CPCs to synthesize matrix metalloproteinases that degraded collagen proteins, forming tunnels within the fibrotic tissue during their migration across the scarred myocardium. New myocytes had a 2n karyotype and possessed 2 sex chromosomes, excluding cell fusion. Clinically, CPCs represent an ideal candidate cell for cardiac repair in patients with chronic heart failure. CPCs may be isolated from myocardial biopsies and, following their expansion in vitro, administered back to the same patients avoiding the adverse effects associated with the use of nonautologous cells. Alternatively, growth factors may be delivered locally to stimulate resident CPCs and promote myocardial regeneration. These forms of treatments could be repeated over time to reduce progressively tissue scarring and expand the working myocardium.

Anthracycline Cardiomyopathy Is Mediated by Depletion of the Cardiac Stem Cell Pool and Is Rescued by Restoration of Progenitor Cell Function

Background— Anthracyclines are the most effective drugs available in the treatment of neoplastic diseases; however, they have profound consequences on the structure and function of the heart, which over time cause a cardiomyopathy that leads to congestive heart failure. Methods and Results— Administration of doxorubicin in rats led to a dilated myopathy, heart failure, and death. To test whether the effects of doxorubicin on cardiac anatomy and function were mediated by alterations in cardiac progenitor cells (CPCs), these cells were exposed to the anthracycline, which increased the formation of reactive oxygen species and caused increases in DNA damage, expression of p53, telomere attrition, and apoptosis. Additionally, doxorubicin resulted in cell-cycle arrest at the G2/M transition, which led to a significant decrease in CPC growth. Doxorubicin elicited multiple molecular adaptations; the massive apoptotic death that occurred in CPCs in the presence of anthracycline imposed on the surviving CPC pool the activation of several pathways aimed at preservation of the primitive state, cell division, lineage differentiation, and repair of damaged DNA. To establish whether delivery of syngeneic progenitor cells opposed the progression of doxorubicin cardiotoxicity, enhanced green fluorescent protein–labeled CPCs were injected in the failing myocardium; this treatment promoted regeneration of cardiomyocytes and vascular structures, which improved ventricular performance and rate of animal survival. Conclusions— Our results raise the possibility that autologous CPCs can be obtained before antineoplastic drugs are given to cancer patients and subsequently administered to individuals who are particularly sensitive to the cardiotoxicity of these agents for prevention or management of heart failure.

Formation of large coronary arteries by cardiac progenitor cells.

Coronary artery disease is the most common cause of cardiac failure in the Western world, and to date there is no alternative to bypass surgery for severe coronary atherosclerosis. We report that c-kit-positive cardiac progenitor cells (CPCs) activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection in proximity of the site of occlusion of the left coronary artery in rats, engrafted within the host myocardium forming temporary niches. Subsequently, CPCs divided and differentiated into endothelial cells and smooth muscle cells and, to a lesser extent, into cardiomyocytes. The acquisition of vascular lineages appeared to be mediated by the up-regulation of hypoxia-inducible factor 1α, which promoted the synthesis and secretion of stromal-derived factor 1 from hypoxic coronary vessels. Stromal-derived factor 1 was critical in the conversion of CPCs to the vascular fate. CPCs formed conductive and intermediate-sized coronary arteries together with resistance arterioles and capillaries. The new vessels were connected with the primary coronary circulation, and this increase in vascularization more than doubled myocardial blood flow in the infarcted myocardium. This beneficial effect, together with myocardial regeneration attenuated postinfarction dilated myopathy, reduced infarct size and improved function. In conclusion, locally delivered activated CPCs generate de novo coronary vasculature and may be implemented clinically for restoration of blood supply to the ischemic myocardium.

Spontaneous Calcium Oscillations Regulate Human Cardiac Progenitor Cell Growth

Rationale: The adult heart possesses a pool of progenitor cells stored in myocardial niches, but the mechanisms involved in the activation of this cell compartment are currently unknown. Objective: Ca2+ promotes cell growth raising the possibility that changes in intracellular Ca2+ initiate division of c-kit–positive human cardiac progenitor cells (hCPCs) and determine their fate. Methods and Results: Ca2+ oscillations were identified in hCPCs and these events occurred independently from coupling with cardiomyocytes or the presence of extracellular Ca2+. These findings were confirmed in the heart of transgenic mice in which enhanced green fluorescent protein was under the control of the c-kit promoter. Ca2+ oscillations in hCPCs were regulated by the release of Ca2+ from the endoplasmic reticulum through activation of inositol 1,4,5-triphosphate receptors (IP3Rs) and the reuptake of Ca2+ by the sarco-/endoplasmic reticulum Ca2+ pump (SERCA). IP3Rs and SERCA were highly expressed in hCPCs, whereas ryanodine receptors were not detected. Although Na+-Ca2+ exchanger, store-operated Ca2+ channels and plasma membrane Ca2+ pump were present and functional in hCPCs, they had no direct effects on Ca2+ oscillations. Conversely, Ca2+ oscillations and their frequency markedly increased with ATP and histamine which activated purinoceptors and histamine-1 receptors highly expressed in hCPCs. Importantly, Ca2+ oscillations in hCPCs were coupled with the entry of cells into the cell cycle and 5-bromodeoxyuridine incorporation. Induction of Ca2+ oscillations in hCPCs before their intramyocardial delivery to infarcted hearts was associated with enhanced engraftment and expansion of these cells promoting the generation of a large myocyte progeny. Conclusion: IP3R-mediated Ca2+ mobilization control hCPC growth and their regenerative potential.

The Young Mouse Heart Is Composed of Myocytes Heterogeneous in Age and Function

The recognition that the adult heart continuously renews its myocyte compartment raises the possibility that the age and lifespan of myocytes does not coincide with the age and lifespan of the organ and organism. If this were the case, myocyte turnover would result at any age in a myocardium composed by a heterogeneous population of parenchymal cells which are structurally integrated but may contribute differently to myocardial performance. To test this hypothesis, left ventricular myocytes were isolated from mice at 3 months of age and the contractile, electrical, and calcium cycling characteristics of these cells were determined together with the expression of the senescence-associated protein p16INK4a and telomere length. The heart was characterized by the coexistence of young, aged, and senescent myocytes. Old nonreplicating, p16INK4a-positive, hypertrophied myocytes with severe telomeric shortening were present together with young, dividing, p16INK4a-negative, small myocytes with long telomeres. A class of myocytes with intermediate properties was also found. Physiologically, evidence was obtained in favor of the critical role that action potential (AP) duration and ICaL play in potentiating Ca2+ cycling and the mechanical behavior of young myocytes or in decreasing Ca2+ transients and the performance of senescent hypertrophied cells. The characteristics of the AP appeared to be modulated by the transient outward K+ current Ito which was influenced by the different expression of the K+ channels subunits. Collectively, these observations at the physiological and structural cellular level document that by necessity the heart has to constantly repopulate its myocyte compartment to replace senescent poorly contracting myocytes with younger more efficient cells. Thus, cardiac homeostasis and myocyte turnover regulate cardiac function.

Doxorubicin induces senescence and impairs function of human cardiac progenitor cells

The increasing population of cancer survivors faces considerable morbidity and mortality due to late effects of the antineoplastic therapy. Cardiotoxicity is a major limiting factor of therapy with doxorubicin (DOXO), the most effective anthracycline, and is characterized by a dilated cardiomyopathy that can develop even years after treatment. Studies in animals have proposed the cardiac progenitor cells (CPCs) as the cellular target responsible for DOXO-induced cardiomyopathy but the relevance of these observations to clinical settings is unknown. In this study, the analysis of the DOXO-induced cardiomyopathic human hearts showed that the majority of human CPCs (hCPCs) was senescent. In isolated hCPCs, DOXO triggered DNA damage response leading to apoptosis early after exposure, and telomere shortening and senescence at later time interval. Functional properties of hCPCs, such as migration and differentiation, were also negatively affected. Importantly, the differentiated progeny of DOXO-treated hCPCs prematurely expressed the senescence marker p16INK4a. In conclusion, DOXO exposure severely affects the population of hCPCs and permanently impairs their function. Premature senescence of hCPCs and their progeny can be responsible for the decline in the regenerative capacity of the heart and may represent the cellular basis of DOXO-induced cardiomyopathy in humans.

Mesenchymal stem cell therapy for the treatment of chronic obstructive pulmonary disease

Recent studies have revealed that adult stem cells such as bone marrow-derived cells contribute to lung tissue regeneration and protection, and thus administration of exogenous stem/progenitor cells may be a potent next-generation therapy for COPD. Pathogenesis of COPD is characterized by an upregulation of inflammatory processes leading to irreversible events such as apoptosis of epithelial cells, proteolysis of the terminal air-space and lung extracellular matrix components. The available pharmacological treatments are essentially symptomatic, therefore, there is a need to develop more effective therapeutic strategies. It has been previously demonstrated that transplanted MSC home to the lung in response to lung injury and adopt phenotypes of alveolar epithelial cells, endothelial cells, fibroblasts and bronchial epithelial cells. However, engraftment and differentiation are now felt to be rare occurrences and other mechanisms might be involved and play a more important role. Importantly, MSCs protect lung tissue through suppression of proinflammatory cytokines, and through triggering production of reparative growth factors. Accordingly, it is not clear if and how these cells will be able to repair, to slow or to prevent the disease. This article reviews recent advances in regenerative medicine in COPD and highlights that their potential application although promising and very attractive, are still a far away opinion.