Federico Quaini

Mobilized bone marrow cells repair the infarcted heart, improving function and survival

Attempts to repair myocardial infarcts by transplanting cardiomyocytes or skeletal myoblasts have failed to reconstitute healthy myocardium and coronary vessels integrated structurally and functionally with the remaining viable portion of the ventricular wall. The recently discovered growth and transdifferentiation potential of primitive bone marrow cells (BMC) prompted us, in an earlier study, to inject in the border zone of acute infarcts Lin− c-kitPOS BMC from syngeneic animals. These BMC differentiated into myocytes and vascular structures, ameliorating the function of the infarcted heart. Two critical determinants seem to be required for the transdifferentiation of primitive BMC: tissue damage and a high level of pluripotent cells. On this basis, we hypothesized here that BMC, mobilized by stem cell factor and granulocyte-colony stimulating factor, would home to the infarcted region, replicate, differentiate, and ultimately promote myocardial repair. We report that, in the presence of an acute myocardial infarct, cytokine-mediated translocation of BMC resulted in a significant degree of tissue regeneration 27 days later. Cytokine-induced cardiac repair decreased mortality by 68%, infarct size by 40%, cavitary dilation by 26%, and diastolic stress by 70%. Ejection fraction progressively increased and hemodynamics significantly improved as a consequence of the formation of 15 × 106 new myocytes with arterioles and capillaries connected with the circulation of the unaffected ventricle. In conclusion, mobilization of primitive BMC by cytokines might offer a noninvasive therapeutic strategy for the regeneration of the myocardium lost as a result of ischemic heart disease and, perhaps, other forms of cardiac pathology.

Apoptosis in the Failing Human Heart

BACKGROUND Loss of myocytes is an important mechanism in the development of cardiac failure of either ischemic or nonischemic origin. However, whether programmed cell death (apoptosis) is implicated in the terminal stages of heart failure is not known. We therefore studied the magnitude of myocyte apoptosis in patients with intractable congestive heart failure. METHODS Myocardial samples were obtained from the hearts of 36 patients who underwent cardiac transplantation and from the hearts of 3 patients who died soon after myocardial infarction. Samples from 11 normal hearts were used as controls. Apoptosis was evaluated histochemically, biochemically, and by a combination of histochemical analysis and confocal microscopy. The expression of two proto-oncogenes that influence apoptosis, BCL2 and BAX, was also determined. RESULTS Heart failure was characterized morphologically by a 232-fold increase in myocyte apoptosis and biochemically by DNA laddering (an indicator of apoptosis). The histochemical demonstration of DNA-strand breaks in myocyte nuclei was coupled with the documentation of chromatin condensation and fragmentation by confocal microscopy. All these findings reflect apoptosis of myocytes. The percentage of myocytes labeled with BCL2 (which protects cells against apoptosis) was 1.8 times as high in the hearts of patients with cardiac failure as in the normal hearts, whereas labeling with BAX (which promotes apoptosis) remained constant. The near doubling of the expression of BCL2 in the cardiac tissue of patients with heart failure was confirmed by Western blotting. CONCLUSIONS Programmed death of myocytes occurs in the decompensated human heart in spite of the enhanced expression of BCL2; this phenomenon may contribute to the progression of cardiac dysfunction.

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.

Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy.

It is generally believed that increase in adult contractile cardiac mass can be accomplished only by hypertrophy of existing myocytes. Documentation of myocardial regeneration in acute stress has challenged this dogma and led to the proposition that myocyte renewal is fundamental to cardiac homeostasis. Here we report that in human aortic stenosis, increased cardiac mass results from a combination of myocyte hypertrophy and hyperplasia. Intense new myocyte formation results from the differentiation of stem-like cells committed to the myocyte lineage. These cells express stem cell markers and telomerase. Their number increased >13-fold in aortic stenosis. The finding of cell clusters with stem cells making the transition to cardiogenic and myocyte precursors, as well as very primitive myocytes that turn into terminally differentiated myocytes, provides a link between cardiac stem cells and myocyte differentiation. Growth and differentiation of these primitive cells was markedly enhanced in hypertrophy, consistent with activation of a restricted number of stem cells that, through symmetrical cell division, generate asynchronously differentiating progeny. These clusters strongly support the existence of cardiac stem cells that amplify and commit to the myocyte lineage in response to increased workload. Their presence is consistent with the notion that myocyte hyperplasia significantly contributes to cardiac hypertrophy and accounts for the subpopulation of cycling myocytes.

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.

Evidence for Human Lung Stem Cells

BACKGROUND Although progenitor cells have been described in distinct anatomical regions of the lung, description of resident stem cells has remained elusive. METHODS Surgical lung-tissue specimens were studied in situ to identify and characterize human lung stem cells. We defined their phenotype and functional properties in vitro and in vivo. RESULTS Human lungs contain undifferentiated human lung stem cells nested in niches in the distal airways. These cells are self-renewing, clonogenic, and multipotent in vitro. After injection into damaged mouse lung in vivo, human lung stem cells form human bronchioles, alveoli, and pulmonary vessels integrated structurally and functionally with the damaged organ. The formation of a chimeric lung was confirmed by detection of human transcripts for epithelial and vascular genes. In addition, the self-renewal and long-term proliferation of human lung stem cells was shown in serial-transplantation assays. CONCLUSIONS Human lungs contain identifiable stem cells. In animal models, these cells participate in tissue homeostasis and regeneration. They have the undemonstrated potential to promote tissue restoration in patients with lung disease. (Funded by the National Institutes of Health.).

Myocyte Turnover in the Aging Human Heart

Rationale: The turnover of cardiomyocytes in the aging female and male heart is currently unknown, emphasizing the need to define human myocardial biology. Objective: The effects of age and gender on the magnitude of myocyte regeneration and the origin of newly formed cardiomyocytes were determined. Methods and Results: The interaction of myocyte replacement, cellular senescence, growth inhibition, and apoptosis was measured in normal female (n=32) and male (n=42) human hearts collected from patients 19 to 104 years of age who died from causes other than cardiovascular diseases. A progressive loss of telomeric DNA in human cardiac stem cells (hCSCs) occurs with aging and the newly formed cardiomyocytes inherit short telomeres and rapidly reach the senescent phenotype. Our data provide novel information on the superior ability of the female heart to sustain the multiple variables associated with the development of the senescent myopathy. At all ages, the female heart is equipped with a larger pool of functionally competent hCSCs and younger myocytes than the male myocardium. The replicative potential is higher and telomeres are longer in female hCSCs than in male hCSCs. In the female heart, myocyte turnover occurs at a rate of 10%, 14%, and 40% per year at 20, 60, and 100 years of age, respectively. Corresponding values in the male heart are 7%, 12%, and 32% per year, documenting that cardiomyogenesis involves a large and progressively increasing number of parenchymal cells with aging. From 20 to 100 years of age, the myocyte compartment is replaced 15 times in women and 11 times in men. Conclusions: The human heart is a highly dynamic organ regulated by a pool of resident hCSCs that modulate cardiac homeostasis and condition organ aging.

Diabetes Impairs Hematopoietic Stem Cell Mobilization by Altering Niche Function

Impaired mobilization of hematopoietic stem cells in diabetic mice is due to sympathetic nervous system dysregulation of CXCL12 distribution. Boosting Stem Cell Mobilization Transplantation of hematopoietic stem cells (HSCs) from the bone marrow is a successful approach for treating blood diseases and certain cancers. Usually, the patient’s own (autologous) HSCs are used for transplant, but in some patients, their HSCs cannot be mobilized in sufficient numbers using the growth factor G-CSF (granulocyte colony-stimulating factor) to enable a successful transplant. In a new study, Ferraro and colleagues set out to discover the causes of this poor HSC mobilization. The investigators discovered by analyzing data from a number of bone marrow transplant patients that patients with diabetes showed poorer mobilization of HSCs in response to G-CSF than did those patients who did not have diabetes. The authors then confirmed in mouse models of type 1 and type 2 diabetes that HSCs were poorly mobilized from the bone marrow in response to G-CSF in these mice but not healthy control animals. The authors discovered that there was a defect in the bone marrow microenvironment of the diabetic mice rather than a problem with the HSCs themselves. Specifically, in diabetic (but not control) mice, the researchers observed mislocalization of HSCs in the bone marrow and an increase in the number of perivascular sympathetic nerve fibers in the niche with a concomitant inability of bone marrow mesenchymal stem cells to down-modulate production of the chemokine CXCL12 (a molecule known to mediate HSC localization). Finally, the authors were able to overcome the defect in HSC mobilization using a clinically approved drug called AMD3100 that interrupts the interaction of CXCL12 with its receptor CXCR4. The authors suggest that AMD3100 could be used to boost HSC mobilization in diabetic patients who require a bone marrow transplant. Success with transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) in patients depends on adequate collection of these cells after mobilization from the bone marrow niche by the cytokine granulocyte colony-stimulating factor (G-CSF). However, some patients fail to achieve sufficient HSPC mobilization. Retrospective analysis of bone marrow transplant patient records revealed that diabetes correlated with poor mobilization of CD34+ HSPCs. In mouse models of type 1 and type 2 diabetes (streptozotocin-induced and db/db mice, respectively), we found impaired egress of murine HSPCs from the bone marrow after G-CSF treatment. Furthermore, HSPCs were aberrantly localized in the marrow niche of the diabetic mice, and abnormalities in the number and function of sympathetic nerve termini were associated with this mislocalization. Aberrant responses to β-adrenergic stimulation of the bone marrow included an inability of marrow mesenchymal stem cells expressing the marker nestin to down-modulate the chemokine CXCL12 in response to G-CSF treatment (mesenchymal stem cells are reported to be critical for HSPC mobilization). The HSPC mobilization defect was rescued by direct pharmacological inhibition of the interaction of CXCL12 with its receptor CXCR4 using the drug AMD3100. These data suggest that there are diabetes-induced changes in bone marrow physiology and microanatomy and point to a potential intervention to overcome poor HSPC mobilization in diabetic patients.

Diabetes Mellitus Induces Bone Marrow Microangiopathy

Objective—The impact of diabetes on the bone marrow (BM) microenvironment was not adequately explored. We investigated whether diabetes induces microvascular remodeling with negative consequence for BM homeostasis. Methods and Results—We found profound structural alterations in BM from mice with type 1 diabetes with depletion of the hematopoietic component and fatty degeneration. Blood flow (fluorescent microspheres) and microvascular density (immunohistochemistry) were remarkably reduced. Flow cytometry verified the depletion of MECA-32+ endothelial cells. Cultured endothelial cells from BM of diabetic mice showed higher levels of oxidative stress, increased activity of the senescence marker &bgr;-galactosidase, reduced migratory and network-formation capacities, and increased permeability and adhesiveness to BM mononuclear cells. Flow cytometry analysis of lineage− c-Kit+ Sca-1+ cell distribution along an in vivo Hoechst-33342 dye perfusion gradient documented that diabetes depletes lineage− c-Kit+ Sca-1+ cells predominantly in the low-perfused part of the marrow. Cell depletion was associated to increased oxidative stress, DNA damage, and activation of apoptosis. Boosting the antioxidative pentose phosphate pathway by benfotiamine supplementation prevented microangiopathy, hypoperfusion, and lineage− c-Kit+ Sca-1+ cell depletion. Conclusion—We provide novel evidence for the presence of microangiopathy impinging on the integrity of diabetic BM. These discoveries offer the framework for mechanistic solutions of BM dysfunction in diabetes.

Nerve growth factor promotes cardiac repair following myocardial infarction

Rationale: Nerve growth factor (NGF) promotes angiogenesis and cardiomyocyte survival, which are both desirable for postinfarction myocardial healing. Nonetheless, the NGF potential for cardiac repair has never been investigated. Objective: To define expression and localization of NGF and its high-affinity receptor TrkA (tropomyosin-related receptor A) in the human infarcted heart and to investigate the cardiac roles of both endogenous and engineered NGF using a mouse model of myocardial infarction (MI). Methods and Results: Immunostaining for NGF and TrkA was performed on heart samples from humans deceased of MI or unrelated pathologies. To study the post-MI functions of endogenous NGF, a NGF-neutralizing antibody (Ab-NGF) or nonimmune IgG (control) was given to MI mice. To investigate the NGF therapeutic potential, human NGF gene or control (empty vector) was delivered to the murine periinfarct myocardium. Results indicate that NGF is present in the infarcted human heart. Both cardiomyocytes and endothelial cells (ECs) possess TrkA, which suggests NGF cardiovascular actions in humans. In MI mice, Ab-NGF abrogated native reparative angiogenesis, increased EC and cardiomyocyte apoptosis and worsened cardiac function. Conversely, NGF gene transfer ameliorated EC and cardiomyocyte survival, promoted neovascularization and improved myocardial blood flow and cardiac function. The prosurvival/proangiogenic Akt/Foxo pathway mediated the therapeutic benefits of NGF transfer. Moreover, NGF overexpression increased stem cell factor (the c-kit receptor ligand) expression, which translated in higher myocardial abundance of c-kitpos progenitor cells in NGF-engineered hearts. Conclusions: NGF elicits pleiotropic beneficial actions in the post-MI heart. NGF should be considered as a candidate for therapeutic cardiac regeneration.