Myocardial cell therapy at the crossroads.
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Animals↗
Biomedical subjects
Bernardo Nadal-Ginard
Publications and source records attributed to Bernardo Nadal-Ginard.
Growth-factor-mediated cardiac stem cell activation in myocardial regeneration.
The concept of an intrinsic regenerative capacity of the adult mammalian myocardium owing to the presence of cardiac stem cells (CSCs) in the atria and ventricles is starting to be accepted by the cardiovascular research community. The identification of this cell population has improved the prospects for developing successful clinical protocols for human myocardial regeneration. In the normal adult myocardium, only a small fraction of CSCs undergo amplification and differentiation to replace the parenchymal cells lost by normal wear and tear. Physiological or pathological stimuli cause substantial activation of CSCs, which is mediated by a paracrine feedback loop between myocytes and CSCs. In response to stress, the myocytes produce growth factors and cytokines, for which CSCs have receptors, and autocrine, self-sustaining activation of growth-factor production is simultaneously triggered in the CSCs. These findings from human and animal studies led us to test whether in situ activation of CSCs by growth factors would be as effective as transplantation of CSCs into the regenerating myocardium after ischemia in an animal model that has relevance to humans. In a porcine model, we produced extensive and functionally relevant myocardial regeneration. Here, we discuss the properties of endogenous myocardial stem cells that might be exploited to produce clinical myocardial regeneration without the need for cell transplantation.
Heart↗
Myocyte death and renewal: modern concepts of cardiac cellular homeostasis.
The adult mammalian myocardium has a robust intrinsic regenerative capacity because of the presence of cardiac stem cells (CSCs). Despite being mainly composed of terminally differentiated myocytes that cannot re-enter the cell cycle, the heart is not a postmitotic organ and maintains some capacity to form new parenchymal cells during the lifespan of the organism. Myocyte death and formation of new myocytes by the CSCs are the two processes that enable this organ to maintain a proper and uninterrupted cardiac output from birth to adulthood and into old age. CSCs are activated in response to pathological or physiological stimuli, whereby they enter the cell cycle and differentiate into new myocytes (and vessels) that significantly contribute to changes in myocardial mass. The future of regenerative cardiovascular medicine is arguably dependent on our success in dissecting the biology and mechanisms regulating the number, growth, differentiation, and aging of CSCs. This information will generate the means to manipulate CSC growth, survival, and differentiation and, therefore, will provide the tools for the design of more physiologically relevant clinical regeneration protocols. In this article, we review the developments in cardiac cell biology that might, in our opinion, have a broad impact on cardiovascular medicine.
Adult↗
Bone marrow cells have a potent anti-ischemic effect against myocardial cell death in humans.
OBJECTIVE: We sought to elucidate whether bone marrow cells ameliorate the outcomes of myocardial ischemia by reduction of cell death and to investigate whether the benefit is mediated by activation of intracellular kinases. METHODS: Muscles from the right atrial appendage of patients were subjected to 90 minutes of normothermic simulated ischemia followed by 120 minutes of reoxygenation. Bone marrow cells from the same patients were co-incubated (10(5) cells per milligram of tissue) with the muscles during the entire experimental period. Some groups were treated with the protein kinase C inhibitor chelerythrine (10 micromol/L) or the p38 mitogen-activated protein kinase inhibitor SB203580 (10 micromol/L). Creatine kinase released into the media during the reoxygenation period was measured (international units per milligram of wet tissue), cell death by necrosis was assessed by propidium iodide, and cell death by apoptosis was assessed by deoxyuride-5'-triphosphate biotin nick end labeling (percentage of aerobic control values). RESULTS: Creatine kinase release was significantly reduced (from 1.30 IU/mg wet tissue +/- 0.11 to 0.33 IU/mg wet tissue +/- 0.06; P < .05), and cell death by necrosis and apoptosis was abolished by bone marrow cells (from 30.1% +/- 7.3% and 28.1% +/- 3.9% to -5.6% +/- 5.1% and 3.7% +/- 5.0%, respectively; P < .05), an effect that was reversed by chelerythrine (13.4% +/- 4.4% and 24.6% +/- 8.2%, respectively) and by SB203580 (20.1% +/- 2.4% and 19.5% +/- 5.7%, respectively). CONCLUSIONS: Bone marrow cells have a potent effect against cell death of the human myocardium in the acute phase of ischemia that may explain, at least in part, the improvement in cardiac function and the reduction in infarct size seen when bone marrow cells are injected after a myocardial infarction. These findings may have important clinical implications to optimize cell therapy with bone marrow cells. In addition, the identification that the anti-ischemic effect of bone marrow cells is mediated by the kinases protein kinase C and p38 mitogen-activated protein kinase is also clinically relevant; it suggests that some of the beneficial effect of bone marrow cells can be obtained by the activation of intracellular signaling molecules, without the need for cell injection.
Alkaloids↗
Resident human cardiac stem cells: role in cardiac cellular homeostasis and potential for myocardial regeneration.
Current treatments for myocardial infarction have significantly reduced the acute mortality of ischemic cardiomyopathy. This reduction has resulted in the survival of a large cohort of patients left with a significant 'myocyte deficit'. Once this deficit leads to heart failure there is no available therapy to improve long-term cardiac function. Recent developments in stem cell biology have focused on the possibility of regenerating contractile myocardial tissue. Most of these approaches have entailed the transplantation of exogenous cardiac-regenerating cells. Recently, we and others have reported that the adult mammalian myocardium, including that in humans, contains a small pool of cardiac stem and progenitor cells (CSCs) that can replenish the cardiomyocyte population and, in some cases, the coronary microcirculation. The human CSCs (hCSCs) are involved in maintaining myocardial cell homeostasis throughout life and participate in remodeling in cardiac pathology. They can be isolated, propagated and cloned. The progeny of a single cell clone differentiates in vitro and in vivo into myocytes, smooth muscle and endothelial cells. Surprisingly, in response to different forms of stress, hCSCs acquire a senescent, dysfunctional phenotype. Strikingly, these nonfunctional CSCs constitute around 50% of the total CSC pool in older individuals-those most likely to be candidates for hCSC-based myocardial regeneration. Therefore, the challenge to develop clinically effective therapies of myocardial regeneration is twofold: to produce the activation of the hCSCs in situ in order to obviate the need for cell transplantation, and to elucidate the mechanisms responsible for hCSC senescence in order to prevent or reverse its development.
Aging↗
Resident progenitors and bone marrow stem cells in myocardial renewal and repair.
Although cardiac transplantation is still the treatment of choice for end-stage heart disease, the side effects derived from the use of immunosuppressants and the limited availability of donors have prompted the search for alternative therapeutic strategies. Among other possibilities, cell transplantation approaches have recently emerged as new alternatives to stimulate myocardial regeneration. These approaches are mainly based on the increasing number of reports documenting the plasticity of stem cells of various origins, particularly the ability of several types of embryonic and adult stem cells to give rise to cardiomyocytes. Unprecedented in the field of 'translational research' and based on the urgent need for alternative therapies, the promising results obtained with animal models have been quickly transferred to the clinical arena, where numerous small pilot studies using different cell types are already ongoing and/or have reported promising results. Nevertheless, the lack of randomization, the variability and small size of the treated cohorts and the use of mixed populations of cells have often clouded the significance and prevented a mechanistic interpretation of the results. Here, we briefly review the use of bone-marrow-derived and cardiac-derived stem/progenitor cells in myocardial regeneration studies and discuss their significance for the future of the field of myocardial regeneration.
Animals↗
[Cardiovascular regenerative medicine at the crossroads. Clinical trials of cellular therapy must now be based on reliable experimental data from animals with characteristics similar to human's].
It is now over 4 years since early reports of murine models raised high expectations that bone marrow cell transplantation to the postischemic myocardium could produce physiologically significant myocardial regeneration. In quick succession, a flurry of publications documented the capacity of a variety of other types of adult cell to produce similar results. These publications were all controversial from the start because none addressed the mechanisms involved in the differentiation of transplanted cells. In addition, each report raised at least as many questions as it answered. Despite these obvious weaknesses, the first phase-I clinical trials were started immediately without any further animal experimentation. Today the results of more than a dozen trials are already in the public domain but we still do not have a single piece of solid data documenting whether any of the approaches used is capable of regenerating contractile cells in the human myocardium. This is one of the main reasons why the controversy over the effectiveness of this therapeutic approach is becoming increasingly heated. Moreover, skepticism about the efficacy, and even the feasibility, of inducing clinically relevant myocardial regeneration has increased to the point where it threatens the future of this nascent field. The present situation in myocardial generation contrasts sharply with that in neural regeneration. Although there is a solid and extensive body of knowledge on the origin, phenotype, and regulatory mechanisms of neural stem cells, the first clinical trials have only recently been started. To move this field forward it is necessary to distinguish between the procedures needed to establish proof-of-concept and those that have the potential for widespread clinical application. In addition, the technique must be implemented in such a way that it continues to add to existing knowledge. It is our belief that, if the necessary information is to be acquired, we need: a) significantly more extensive experimental data from animals whose anatomical and physiological characteristics are similar to human's, including data on, for example, dose-effect relationships, the best form of administration, and the duration of therapeutic responses; and b) better understanding of the molecular mechanisms that determine whether cardiac stem cells and transplanted cells will either remain as stem cells or differentiate. In summary, if we are to progress systematically in this area, we need better understanding of myocardial biology. Without it, we run the risk of holding back the field for decades, as happened with the first human heart transplants and with trials of gene therapy.
Adult Stem Cells↗
Cardiac stem and progenitor cell biology for regenerative medicine.
Stem cell therapy is a new and promising treatment of heart disease. However, the race is still on to find the "best" cell to reconstitute the myocardium and improve function after myocardial damage. The recent discovery in the adult mammalian myocardium of a small cell population with the phenotype, behavior, and regenerative potential of cardiac stem and progenitor cells has proposed these cells as the most appropriate for cell therapy. The existence of these cells has provided an explanation for the hitherto unexplained existence of a subpopulation of immature cycling myocytes in the adult myocardium. These findings have placed the heart squarely among other organs with regenerative potential despite the fact that the working myocardium is mainly constituted of terminally differentiated cells. Although CSCs (cardiac cells proven to have stem and/or progenitor characteristics) can be isolated and amplified in vitro or stimulated to differentiate in situ, it has become reasonable to exploit this endogenous regenerative potential to replace the lost muscle with autologous functional myocardium. Therefore, it is imperative to obtain a better understanding of the biology and regenerative potential of the endogenous CSCs. This will enable us to design better protocols for the regeneration of functional contractile mass after myocardial injury.
Animals↗
Cardiac stem cells and myocardial regeneration.
Until recently, the accepted paradigm considered the adult mammalian heart a post-mitotic organ without intrinsic regenerative capacity where neither myocyte death nor new myocyte formation played any role in its homeostasis and could be safely ignored. We have recently identified in the adult mammalian myocardium a small cell population expressing surface antigens commonly associated with a variety of stem cells. These cells have the behaviour and potential of bonafide cardiac stem cells (CSCs): they are clonogenic, self-renewing and multipotent. Their presence has identified myocyte death and myocyte renewal as the two sides of the proverbial coin of cardiac homeostasis. Myocyte renewal depends on the differentiation of the CSCs into immature myocytes that divide two to four times before becoming terminally differentiated. Both in vivo and in vitro the progeny of a single CSC can generate the three major cell types of the myocardium: myocytes, smooth muscle and endothelial vascular cells. More interestingly, when directly injected or activated with growth factors in the post-ischaemic myocardium, these cells are able to reconstitute a functional ventricular wall. Thus, although in the adult heart most cardiac myocytes are permanently withdrawn from the cell cycle, the heart has an intrinsic regenerative potential and it is not a terminally differentiated organ.
Animals↗
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.
Animals↗
Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression.
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.
Aging↗
Myocyte growth in the failing heart.
Adult ventricular myocytes can undergo mitotic division, resulting in an increase in the aggregate number of cells in the heart. The improvement in the methodological approach to the analysis of tissue sections by immunostaining and confocal microscopy has defeated the dogma that myocyte regeneration cannot occur in the adult heart. Most importantly, primitive and progenitor cells have been identified in the human heart. These cells express telomerase and have the capability of undergoing lineage commitment and rapid cell division, expanding significantly the contracting ventricular myocardium. These cell populations possess all the molecular components regulating the entry and progression through the cell cycle, karyokinesis, and cytokinesis. The recognition that myocyte hypertrophy and regeneration, as well as myocyte necrosis and apoptosis, occur in cardiac diseases has contributed to enhancing our understanding of the plasticity of the human heart.
Apoptosis↗
Adult cardiac stem cells are multipotent and support myocardial regeneration.
The notion of the adult heart as terminally differentiated organ without self-renewal potential has been undermined by the existence of a subpopulation of replicating myocytes in normal and pathological states. The origin and significance of these cells has remained obscure for lack of a proper biological context. We report the existence of Lin(-) c-kit(POS) cells with the properties of cardiac stem cells. They are self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells. When injected into an ischemic heart, these cells or their clonal progeny reconstitute well-differentiated myocardium, formed by blood-carrying new vessels and myocytes with the characteristics of young cells, encompassing approximately 70% of the ventricle. Thus, the adult heart, like the brain, is mainly composed of terminally differentiated cells, but is not a terminally differentiated organ because it contains stem cells supporting its regeneration. The existence of these cells opens new opportunities for myocardial repair.
Animals↗
Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure.
Chronological myocardial aging is viewed as the inevitable effect of time on the functional reserve of the heart. Cardiac failure in elderly patients is commonly interpreted as an idiopathic or secondary myopathy superimposed on the old heart independently from the aging process. Thus, aged diseased hearts were studied to determine whether cell regeneration was disproportionate to the accumulation of old dying cells, leading to cardiac decompensation. Endomyocardial biopsies from 19 old patients with a dilated myopathy were compared with specimens from 7 individuals of similar age and normal ventricular function. Ten patients with idiopathic dilated cardiomyopathy were also analyzed to detect differences with aged diseased hearts. Senescent cells were identified by the expression of the cell cycle inhibitor p16INK4a and cell death by hairpin 1 and 2. Replication of primitive cells and myocytes was assessed by MCM5 labeling, myocyte mitotic index, and telomerase function. Aged diseased hearts had moderate hypertrophy and dilation, accumulation of p16INK4a positive primitive cells and myocytes, and no structural damage. Cell death markedly increased and occurred only in cells expressing p16INK4a that had significant telomeric shortening. Cell multiplication, mitotic index and telomerase increased but did not compensate for cell death or prevented telomeric shortening. Idiopathic dilated cardiomyopathy had severe hypertrophy and dilation, tissue injury, and minimal level of p16INK4a labeling. In conclusion, telomere erosion, cellular senescence, and death characterize aged diseased hearts and the development of cardiac failure in humans.
Aged↗
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.
Cardiomegaly↗
Primitive cells and tissue regeneration.
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Animals↗
Myocyte death, growth, and regeneration in cardiac hypertrophy and failure.
The accepted paradigm considers the adult mammalian heart as a postmitotic organ, which possesses a relatively constant number of myocytes from shortly after birth to adulthood and senescence. This notion is questioned by the demonstration that although most adult myocytes are terminally differentiated, there is a small and continuously renewed subpopulation of cycling myocytes produced by the differentiation of cardiac stem-like cells. Myocyte death and myocyte regeneration are introduced as major determinants of cardiac homeostasis and alterations of ventricular anatomy and function in physiological and pathological states. The possibility of reconstituting dead myocardium by stem-like cells is advanced and proposed as a major area of future research.
Animals↗