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Monitoring Stem Cell Research


Table of Contents

The President's Council on Bioethics
Washington, D.C.
January 2004
www.bioethics.gov


Pre-Publication Version
Appendix M

Potential Use of Cellular Therapy for Patients with Heart Disease

Silviu Itescu M.D.
Departments of Medicine and Surgery,
Columbia University, New York, NY

Summary

Congestive heart failure remains a major public health problem, and is frequently the end result of cardiomyocyte apoptosis and fibrous replacement after myocardial infarction, a process referred to as left ventricular remodelling. Cardiomyocytes undergo terminal differentiation soon after birth, and are generally considered to irreversibly withdraw from the cell cycle.  In response to ischemic insult, adult cardiomyocytes undergo cellular hypertrophy, nuclear ploidy, and a high degree of apoptosis.  A small number of human cardiomyocytes retain the capacity to proliferate and regenerate in response to ischemic injury, however whether these cells are derived from a resident pool of cardiomyocyte stem cells or from a renewable source of circulating bone marrow-derived stem cells that home to the damaged myocardium is at present not known.  Replacement and regeneration of functional cardiac muscle after an ischemic insult to the heart could be achieved by either stimulating proliferation of endogenous mature cardiomyocytes or resident cardiac stem cells, or by implanting exogenous donor-derived or allogeneic cells such as fetal or embryonic cardiomyocyte precursors, bone marrow-derived mesenchymal stem cells, or skeletal myoblasts.  The newly formed cardiomyocytes must integrate precisely into the existing myocardial wall in order to augment synchronized contractility and avoid potentially life-threatening alterations in the electrical conduction of the heart.  A major impediment to survival of the implanted cells is altered immunogenicity by prolonged ex vivo culture conditions. In addition, concurrent myocardial revascularization is required to ensure viability of the repaired region and prevent further scar tissue formation.  Human adult bone marrow contains endothelial precursors which resemble embryonic angioblasts and can be used to induce infarct bed neovascularization after experimental myocardial infarction.  This results in protection of cardiomyocytes against apoptosis, induction of cardiomyocyte proliferation and regeneration, long-term salvage and survival of viable myocardium, prevention of left ventricular remodelling and sustained improvement in cardiac function.  It is reasonable to anticipate that cell therapy strategies for ischemic heart disease will need to incorporate (1) a renewable source of proliferating, functional cardiomyocytes, and (2) angioblasts to generate a network of capillaries and larger size blood vessels for supply of oxygen and nutrients to both the chronically ischemic endogenous myocardium and to the newly-implanted cardiomyocytes.

Introduction

Congestive heart failure remains a major public health problem, with recent estimates indicating that end-stage heart failure with two-year mortality rates of 70-80% affects over 60,000 patients in the US each year1.  In Western societies heart failure is primarily the consequence of previous myocardial infarction2.  As new modalities have emerged which have enabled significant reduction in early mortality from acute myocardial infarction, affecting over 1 million new patients in the US annually, there has been a paradoxical increase in the incidence of post-infarction heart failure among the survivors.  Current therapy of heart failure is limited to the treatment of already established disease and is predominantly pharmacological in nature, aiming primarily to inhibit the neurohormonal axis that results in excessive cardiac activation through angiotensin- or norepinephrine-dependent pathways. For patients with end-stage heart failure treatment options are extremely limited, with less than 3000 being offered cardiac transplants annually due to the severely limited supply of donor organs3,4, and implantable left ventricular assist devices (LVADs) being expensive, not proven for long-term use, and associated with significant complications5-7. Clearly, development of approaches that prevent heart failure after myocardial infarction would be preferable to those that simply ameliorate or treat already established disease.

Heart Failure After Myocardial Infarction Results From Progressive Ventricular Remodelling.   Heart failure after myocardial infarction occurs as a result of a process termed myocardial remodelling. This process is characterized by myocyte apoptosis, cardiomyocyte replacement by fibrous tissue deposition in the ventricular wall8-10, progressive expansion of the initial infarct area and dilation of the left ventricular lumen11,12.  Another integral component of the remodelling process appears to be the development of neoangiogenesis within the myocardial infarct scar13,14, a process requiring activation of latent collagenase and other proteinases15.  Under normal circumstances, the contribution of neoangiogenesis to the infarct bed capillary network is insufficient to keep pace with the tissue growth required for contractile compensation and is unable to support the greater demands of the hypertrophied, but viable, myocardium.  The relative lack of oxygen and nutrients to the hypertrophied myocytes may be an important etiological factor in the death of otherwise viable myocardium, resulting in progressive infarct extension and fibrous replacement.  Since late reperfusion of the infarct vascular bed in both humans and experimental animal models significantly benefits ventricular remodelling and survival16-18, we have postulated that methods to successfully augment vascular bed neovascularization might improve cardiac function by preventing loss of hypertrophied, but otherwise viable, cardiac myocytes.

Inability Of Damaged Myocardium To Undergo Repair Due To Cell Cycle Arrest Of Adult Cardiomyocytes.  Cardiomyocytes undergo terminal differentiation soon after birth, and are thought by most investigators to irreversibly withdraw from the cell cycle. Analysis of cardiac myocyte growth during early mammalian development indicates that cardiac myocyte DNA synthesis occurs primarily in utero, with proliferating cells decreasing from 33% at mid-gestation to 2% at birth19. While ventricular karyokinesis and cytokinesis are coupled during fetal growth, resulting in increases in mononucleated cardiac myocytes, karyokinesis occurs in the absence of cytokinesis for a transient period during the post-natal period, resulting in binucleation of ventricular myocytes without an overall increase in cell number.  A similar dissociation between karyokinesis and cytokinesis characterizes the primary adult mammalian cardiac response to ischemia, resulting in myocyte hypertrophy and increase in nuclear ploidy rather than myocyte hyperplasia20,21.  Moreover, in parallel with an inability to progress through cell cycle, ischemic adult cardiomyocytes undergo a high degree of apoptosis.

When cells proliferate, the mitotic cycle progression is tightly regulated by an intricate network of positive and negative signals. Progress from one phase of the cell cycle to the next is controlled by the transduction of mitogenic signals to cyclically expressed proteins known as cyclins and subsequent activation or inactivation of several members of a conserved family of serine/threonine protein kinases known as the cyclin-dependent kinases (cdks)22. Growth arrest observed with such diverse processes as DNA damage, terminal differentiation, and replicative senescence is due to negative regulation of cell cycle progression by two functionally distinct families of Cdk inhibitors, the Ink4 and Cip/Kip families19.  The cell cycle inhibitory activity of p21Cip1/WAF1 is intimately correlated with its nuclear localization and participation in quaternary complexes of cell cycle regulators by binding to G1 cyclin-CDK through its N-terminal domain and to proliferating cell nuclear antigen (PCNA) through its C-terminal domain 23-26.  The latter interaction blocks the ability of PCNA to activate DNA polymerase, the principal replicative DNA polymerase27. For a growth-arrested cell to subsequently enter an apoptotic pathway requires signals provided by specific apoptotic stimuli in concert with cell-cycle regulators.  For example, caspase-mediated cleavage of p21, together with upregulation of cyclin A-associated cdk2 activity, have been shown to be critical steps for induction of cellular apoptosis by either deprivation of growth factors28 or hypoxia of cardiomyocytes29.

Throughout life, a mixture of young and old cells is present in the normal myocardium. Although most myocytes seem to be terminally differentiated, there is a fraction of younger myocytes (15-20%) that retains the capacity to replicate30. Moreover, recent observations have suggested that some human ventricular cardiomyocytes also have the capacity to proliferate and regenerate in response to ischemic injury31,32.  The dividing myocytes can be identified on the basis of immunohistochemical staining of proliferating nuclear structures such as Ki67 and cell surface expression of specific surface markers, including c-kit (CD117).  Whether these cells are derived from a resident pool of cardiomyocyte stem cells or are derived from a renewable source of circulating bone marrow-derived stem cells that home to the damaged myocardium remains to be determined.   More importantly, the signals required for homing, in situ expansion and differentiation of these cells are, at present, unknown.  Gaining an understanding of these issues would open the possibility of manipulating the biology of endogenous cardiomyocytes in order to augment the healing process after myocardial ischemia.

Strategies For The Use Of Cellular Therapy To Improve Myocardial Function. Replacement and regeneration of functional cardiac muscle after an ischemic insult to the heart could be achieved by either stimulating proliferation of endogenous mature cardiomyocytes or resident cardiac stem cells, or by implanting exogenous donor-derived or allogeneic cardiomyocytes.  The newly formed cardiomyocytes must integrate precisely into the existing myocardial wall in order to augment contractile function of the residual myocardium in a synchronized  manner and avoid alterations in the electrical conduction and syncytial contraction of the heart, potentially resulting in life-threatening consequences. In addition, whatever the source of the cells used, it is likely that concurrent myocardial revascularization must also occur in order to ensure viability of the repaired region and prevent further scar tissue formation.  The following section discusses various methods of using cellular therapies to replace damaged myocardium or re-initiate mitosis in mature endogenous cardiomyocytes, including transplanted bone marrow-derived cardiomyocyte or endothelial precursors, fetal cardiomyocytes, and skeletal myoblasts.

Potential Role For Bone Marrow-Derived Or Embryonic Cardiomyocyte Lineage Stem Cells In Myocardial Repair/Regeneration.  Over the past several years, a number of studies have suggested that stem cells can be used to generate cardiomyocytes ex vivo for potential use in a range of cardiovascular diseases33-37.  Multipotent bone marrow-derived mesenchymal stem cells have been identified in adult murine and human bone marrow functionally by their ability to differentiate to lineages of diverse mesenchymal tissues, including bone, cartilage, fat, tendon, and both skeletal and cardiac muscle36, and phenotypically by their expression of specific surface markers and lack of hematopoietic lineage markers such as CD34 or CD4535.   It is well established that murine embryonic stem (ES) cells can give rise to cardiomyocytes in vitro and in vivo38,39.  Recently, Kehat et al. were able to demonstrate that human embryonic stem cells can also differentiate in vitro into cells with characteristics of cardiomyocytes37.  However, there are striking differences in the human and murine stem cell models, and this needs to be taken into account when extrapolating results of mouse experiments to the human condition.  For example, human ES cells have a very low efficiency of differentiation to cardiomyocytes compared with murine ES cells, and a considerably slower time course (a median of 11 days vs 2 days).  Whether these differences reflect true variations between species, or differences in the experimental protocols, remains to be determined.

Irrespective whether the cardiomyocyte lineage stem cell precursors are obtained from adult bone marrow or embryonic sources, the newly generated cardiomyocytes appear to resemble normal cardiomyocytes in terms of phenotypic properties, such as expression of actinin, desmin and troponin I, and function, including positive and negative chronotropic regulation of contractility by pharmacological agents and production of vasoactive factors such as atrial and brain natriuretic peptides.  However, in vivo evidence for functional cardiac improvement following transplantation of adult bone marrow-derived or ES-derived cardiomyocytes has been exceedingly difficult to show to date.  In part this may be because the signals required for cardiomyocyte differentiation and functional regulation are complex and poorly understood.  For example, phenotypic and functional differentiation of mesenchymal stem cells to cardiomyocyte lineage cells in vitro requires culture with exogenously added 5-azacytidine33,34.  Alternatively, the poor functional data obtained to date may reflect immune-mediated rejection of cells which have been modified during the ex vivo culture process or poor viability due to the lack of a sufficient vascular supply to the engrafted cells (see below).

Potential Role For Autologous Skeletal Myoblasts In Myocardial Repair. An alternative approach to replacing damaged myocardium involves the use of autologous skeletal myoblasts40. The procedure involves harvesting a patient's skeletal muscle cells, expanding the cells in a laboratory, and re-injecting the cells into the patient's heart.  Perceived advantages of the approach include ease of access to the cellular source, the fact that immunosuppression is not needed, and the lack of ethical dilemmas associated with the use of allogeneic or embryonic cells.  It has also been argued that using relatively ischemia-resistant skeletal myoblasts rather than cardiomyocytes might enable higher levels of cell engraftment and survival in infarcted regions of the heart, where cardiomyocytes would probably perish41. 

Successful engraftment of autologous skeletal myoblasts into injured myocardium has been reported in multiple animal models of cardiac injury. These studies have demonstrated survival and engraftment of myoblasts into infarcted or necrotic hearts40, differentiation of the myoblasts into striated cells within the damaged myocardium40, and improved myocardial functional performance40,42,43. Other studies have shown that the survival of transplanted myoblasts can be improved by heat shock pretreatment44, and have confirmed that the benefits of skeletal myoblast transfer are additive with those of conventional therapies, such as angiotensin-converting enzyme inhibition45.  More recently, the procedure has been reported anecdotally to result in improved myocardial function in humans46.  On the basis of these preliminary results, clinical trials have begun both in Europe and in the United States.  In addition to demonstrating functional improvement in large, prospective series, questions that remain to be addressed include whether the skeletal myoblasts can make meaningful electromechanical connections to the surrounding endogenous cardiomyocytes through gap junctions, whether the cellular mass will contract in concert, and whether electrical impulses will be transmitted to the myoblast tissue without inducing significant tachyarrhythmias.

Poor Survival Of Cells Transplanted Into Damaged Myocardium After Ex Vivo Culture. A major limitation to successful cellular therapy in animal models of myocardial damage has been the inability of the introduced donor cells to survive in their host environment, whether such transplants have been congenic (analogous to the autologous scenario in humans) or allogeneic.  It has become clear that a major impediment to survival of the implanted cells is the alteration of their immunogenic character by prolonged ex vivo culture conditions.  For example, whereas myocardial implantation of skeletal muscle in the absence of tissue culture does not induce any adverse immune response and results in grafts showing excellent survival for up to a year, injection of cultured isolated (congenic) myoblasts results in a massive and rapid necrosis of donor myoblasts, with over 90% dead within the first hour after injection47-50.  This rapid myoblast death appears to be mediated by host natural killer (NK) cells50 which respond to immunogenic antigens on the transplanted myoblasts altered by exposure to tissue culture conditions48.  It seems likely that a similar mechanism of host NK cell-mediated rejection will apply also to transplanted ES-derived, cultured cardiomyocytes51 since massive death of injected donor cells is recognized as a major problem with transplanted cardiomyocytes, especially in the inflammatory conditions that follow infarction52.  In this regard, the report that cultured mesenchymal stem cells obtained from adult human bone marrow are not rejected on transfer to other species is intriguing53, needs confirmation in humans, and requires detailed investigation into possible tolerogenic mechanisms.

Concomitant Induction Of Vascular Structures Augments Survival and Function Of Cardiomyocyte Precursors.  An additional explanation for the poor survival of transplanted cardiomyocytes or skeletal myoblasts may be that viability and prolonged function of transplanted cells requires an augmented vascular supply.  Whereas many transplanted cardiomyocytes die by apoptosis, cultured cardiomyocytes that incorporate more vascular structures in vivo demonstrate significantly greater survival52.  Moreover, in situations where transplanted cardiomyocyte precursors contained an admixture of cells also giving rise to vascular structures, survival and function of the newly formed cardiomyocytes has been significantly augmented.

In one study, direct injection of whole rat bone marrow into a cryo-damaged heart resulted in neovascularization, cardiomyocyte regeneration and functional improvement34.   More recently, systemic delivery of highly purified bone marrow-derived hematopoietic stem cells in lethally irradiated mice contributed to the formation of both endothelial cells and long-lived cardiomyocytes in ischemic hearts 53.  Most strikingly, significant improvement in cardiac function of mice who had previously undergone LAD ligation was demonstrated after direct myocardial injection of syngeneic bone marrow-derived stem cells, defined on the basis of c-kit (CD117) expression54.  This population of cells contains a mixture of cellular elements in addition to cardiomyocyte precursors, including CD117-positive endothelial progenitors (see below).  These cells were found to proliferate and differentiate into myocytes, smooth muscle cells and endothelial cells, resulting in the partial regeneration of the destroyed myocardium and prevention of ventricular scarring.  Together, these findings raise the intriguing possibility that for long-term in vivo viability and functional integrity of stem cell-derived cardiomyocytes it may be necessary to induce neovascularization by co-administration of endothelial cell progenitors (see below). 

Endothelial Precursors And Formation Of Vascular Structures During Embryogenesis.  In order to develop successful methods for inducing neovascularization of the adult heart, one needs to understand the process of definitive vascular network formation during embryogenesis.  In the pre-natal period, hemangioblasts derived from the human ventral aorta give rise to cellular elements involved in both vasculogenesis, or formation of the primitive capillary network, and hematopoiesis 55,56.  In addition to hematopoietic lineage markers, embryonic hemangioblasts are characterized by expression of the vascular endothelial cell growth factor receptor-2, VEGFR-2, and have high proliferative potential with blast colony formation in response to VEGF57-60.  Under the regulatory influence of various transcriptional and differentiation factors, embryonic hemangioblasts mature, migrate and differentiate to become endothelial lining cells and create the primitive vasculogenic network. The differentiation of embryonic hemangioblasts to pluripotent stem cells and to endothelial precursors appears to be related to co-expression of the GATA-2 transcription factor, since GATA-2 knockout embryonic stem cells have a complete block in definitive hematopoiesis and seeding of the fetal liver and bone marrow61.  Moreover, the earliest precursor of both hematopoietic and endothelial cell lineage to have diverged from embryonic ventral endothelium has been shown to express VEGF receptors as well as GATA-2 and alpha4-integrins62.   Subsequent to capillary tube formation, the newly-created vasculogenic vessels undergo sprouting, tapering, remodelling, and regression under the direction of VEGF, angiopoietins, and other factors, a process termed angiogenesis.  The final component required for definitive vascular network formation to sustain embryonic organogenesis is influx of mesenchymal lineage cells to form the vascular supporting structures such as smooth muscle cells and pericytes.

Characterization Of Endothelial Progenitors, Or Angioblasts, In Human Adult Bone Marrow.  In studies using various animal models of peripheral ischemia a number of groups have shown the potential of adult bone marrow-derived elements to induce neovascularization of ischemic tissues63-69.  In the most successful of these63, bone marrow-derived cells injected directly into the thighs of rats who had undergone ligation of the left femoral artery and vein induced neovascularization and augmented blood flow in the ischemic limb as documented by laser doppler and immunohistochemical analyses.  Although the nature of the bone marrow-derived endothelial progenitors was not precisely identified in these studies, the cumulative reports indicated that this site may be an important source of endothelial progenitors which could be useful for augmenting collateral vessel growth in ischemic tissues, a process termed therapeutic angiogenesis.

In more recent studies, our group has identified such endothelial progenitors in human adult bone marrow70.  By employing both in vitro and in vivo experimental models we have sought to precisely identify the surface characteristics and biological properties of these bone marrow-derived endothelial progenitor cells. Following G-CSF treatment, mobilized mononuclear cells were harvested and CD34+ cells were separated using anti-CD34 mAb coupled to magnetic beads.  90-95% of CD34+ cells co-expressed the hematopoietic lineage marker CD45, 60-80% co-expressed the stem cell factor receptor CD117, and <1% co-expressed the monocyte/macrophage lineage marker CD14.  By quadruple parameter analysis, the VEGFR-2 positive cells within the CD34+CD117bright subset displayed phenotypic characteristics of endothelial progenitors, including co-expression of Tie-2, as well as AC133, but not markers of mature endothelium such as ecNOS, vWF, E-selectin, and ICAM.  Sorting CD34+ cells on the basis of CD117 bright or dim expression demonstrated that GATA-2 mRNA and protein levels were significaantly higher in the CD117bright population, indicating that human adult bone marrow contains cells with an angioblast-like phenotype.

Since the frequency of circulating endothelial cell precursors in animal models has been shown to be increased by either VEGF71 or regional ischemia63-66, phenotypically-defined angioblasts were examined for proliferative responses to VEGF and to factors in ischemic serum. CD117brightGATA-2hiangioblasts demonstrated significantly higher proliferative responses relative to CD117dimGATA-2lo bone marrow-derived cells from the same donor following culture for 96 hours with either VEGF or ischemic serum.  The expanded angioblast population consisted of large blast cells, defined by forward scatter, which continued to express immature markers, including GATA-2 and CD117bright, but not markers of mature endothelial cells, including eNOS or E-selectin, indicating blast proliferation without differentiation under these culture conditions.  However, culture on fibronectin with endothelial growth medium resulted in outgrowth of monolayers with endothelial morphology and functional and phenotypic features characteristic of endothelial cells, including uniform uptake of acetylated LDL, and co-expression of CD34, factor VIII, and eNOS.  Thus, G-CSF treatment of adult humans mobilizes into the peripheral circulation a bone-marrow derived population with phenotypic and functional characteristics of embryonic angioblasts, as defined by specific surface phenotype, high proliferative responses to VEGF and cytokines in ischemic serum, and ability differentiate into endothelial cells by culture in medium enriched with endothelial growth factors.

Human Angioblasts Induce Neovascularization Of The Myocardial Infarct Zone.  Intravenous injection of freshly-obtained human angioblasts into athymic nude rats who had undergone ligation of the left anterior descending (LAD) coronary artery resulted in infarct zone infiltration within 48 hours.  Few human cells were detected in unaffected areas of hearts with regional infarcts or in myocardium of sham-operated rats.  Histologic examination at two weeks post-infarction revealed that injection of human angioblasts was accompanied by a significant increase in infarct zone microvascularity, cellularity, and numbers of capillaries, and by reduction in matrix deposition and fibrosis in comparison to controls. Neovascularization was significantly increased within both the infarct zone and in the peri-infarct rim in rats receiving angioblasts compared with controls receiving saline or other cells which infiltrated the heart (e.g. CD34- cells or saphenous vein endothelial cells, SVEC).  The neovascularization induced by human angioblasts was due to both an increase in capillaries of human origin as well as of rat origin, as defined by monoclonal antibodies with specificity for human or rat CD31 endothelial markers. Capillaries of human origin, defined by co-expression of DiI fluorescence and human CD31, but not rat CD31, accounted for 20-25% of the total myocardial capillary vasculature, and was located exclusively within the central infarct zone of collagen deposition.  In contrast, capillaries of rat origin, as determined by expression of rat, but not human, CD31, demonstrated a distinctively different pattern of localization, being absent within the central zone of collagen deposition and abundant both at the peri-infarct rim between the region of collagen deposition and myocytes, and between myocytes.

Human Angioblasts Protect Hypertrophied Endogenous Cardiomyocytes Against Apoptosis.   By concomitantly staining rat tissues for the myocyte-specific marker desmin and performing DNA end-labeling using the TUNEL technique, temporal examinations demonstrated that the infarct zone neovascularization induced by injection of human angioblasts prevented an eccentrically-extending pro-apoptotic process evident in saline controls.  Thus, at two weeks post-infarction, myocardial tissue of LAD-ligated rats who received saline demonstrated 6-fold higher numbers of apoptotic myocytes compared with that from rats receiving intravenous injections of human angioblasts.  Moreover, these myocytes had distorted appearance and irregular shape.  In contrast, myocytes from LAD-ligated rats who received human angioblasts had regular, oval shape, and were significantly larger than myocytes from control rats. 

Human Angioblasts Induce Sustained Regeneration/ Proliferation Of Endogenous Cardiomyocytes.  In addition to protection of hypertrophied myocytes against apoptosis, human angioblast-dependent neovascularization resulted in a striking induction of regeneration/proliferation of endogenous rat cardiomyocytes at the peri-infarct rim72.  At two weeks after LAD ligation, rats receiving human angioblasts demonstrated numerous "fingers" of cardiomyocytes of rat origin, as determined by expression of rat MHC class I molecules, extending from the peri-infarct region into the infarct zone.  The islands of cardiomyocytes at the peri-infarct rim in animals receiving human angioblasts contained a high frequency of rat myocytes with DNA activity, as determined by dual staining with mAbs reactive against cardiomyocyte-specific troponin I and rat Ki67.  In contrast, in animals receiving saline there was a high frequency of cells with fibroblast morphology and reactivity with rat Ki67, but not troponin I, within the infarct zone.  The number of cardiomyocytes progressing through cell cycle at the peri-infarct region of rats receiving human angioblasts was 40-fold higher than that at sites distal to the infarct, 20-fold higher than that found in non-infarcted hearts, and 5-fold higher than that at the peri-infarct rim of animals receiving saline72.

Neovascularization Of Acutely Ischemic Myocardium By Human Angioblasts Prevents Ventricular Remodelling And Causes Sustained Improvement In Cardiac Function.  By 15 weeks post-infarction, rats receiving human angioblasts demonstrated markedly smaller scar sizes together with increased mass of viable myocardium within the anterior free wall.  Whereas collagen deposition and scar formation extended almost through the entire left ventricular wall thickness in controls, with aneurysmal dilatation and typical EKG abnormalities, the infarct scar extended only to 20-50% of the left ventricular wall thickness in rats receiving CD34+ cells.  Moreover, pathological collagen deposition in the non-infarct zone was markedly reduced in rats receiving CD34+ cells.  At 15 weeks, the mean proportion of scar/normal left ventricular myocardium was 13% in rats receiving CD34+ cells compared with 36-45% for each of the other groups studied (saline, CD34-, SVEC).

Remarkably, by two weeks after injection of human angioblasts, and in a parallel time-frame with the observed neo-vascularization, left ventricular ejection fraction (LVEF) recovered by a mean of 22%.  This effect was long-lived, with LVEF recovering by a mean of 34% at the end of follow-up, 15 weeks post injection. Neither CD34- cells nor SVEC demonstrated similar effects.  At 15 weeks post-infarction, mean cardiac index in rats injected with human angioblasts was only reduced by 26% relative to normal rats, whereas for each of the other groups it was reduced by 48-59%. Together, these results indicate that the neovascularization, reduction in peri-infarct myocyte apoptosis and increase in cardiomyocyte regeneration/proliferation observed at two weeks prevented myocardial replacement with fibrous tissue and caused sustained improvement in myocardial function.

Potential Use Of Angioblasts In Combination With Cardiomyocyte Progenitors For Repair and Regeneration of Ischemic Myocardium.  While increasing capillary density through angioblast-dependent neovascularization is a promising approach for preventing apoptotic death and inducing regeneration of endogenous cardiomyocytes following acute myocardial infarction, the role of angioblast therapy for the treatment of congestive heart failure following chronic ischemia is at present unknown.  Nevertheless, it is reasonable to anticipate that cellular therapies for congestive heart failure due to ischemic cardiomyopathy will need to address two interdependent processes: (1) a renewable source of proliferating, functional cardiomyocytes, and (2) development of a network of capillaries and larger size blood vessels for supply of oxygen and nutrients to both the chronically ischemic, endogenous myocardium and to the newly-implanted cardiomyocytes.  To achieve these endpoints it is likely that co-administration of angioblasts and mesenchymal stem cells will be needed in order to develop regenerating cardiomyocytes, vascular structures, and supporting cells such as pericytes and smooth muscle cells.  Future studies will need to address the timing, relative concentrations, source and route of delivery of each of these cellular populations in animal models of acute and chronic myocardial ischemia.

In addition to synergistic cellular therapies, it is likely that optimal regimens for the treatment of acute and chronically ischemic hearts will require a combined approach employing additional pharmacological strategies.  For example, augmentation in myocardial function might be achieved by combining infusion of human angioblasts and cardiomyocyte progenitors together with beta blockade, ACE inhibition or AT1-receptor blockade to reduce angiotensin II-dependent cardiac fibroblast proliferation and collagen secretion73-76.  Understanding the potential of defined lineages of stem cells or undifferentiated progenitors, and their interactions with pharmacological interventions, will lead to better and more focussed clinical trial designs using each cell type independently or in combination, depending on which particular clinical indication is being targeted.

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ENDNOTES/References

  1. Effects of enalapril on mortality in severe congestive heart failure. Results of the Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS) Trial Study Group. N Engl J Med 1987; 316(23):1429-1435.
  2. Mahon NG, O'Roke C, Codd MB, et al.  Hospital mortality of acute myocardial infarction in the thrombolytic era. Heart 1999;81:478-82.
  3. Hognes JR. In The Artificial Heart: Prototypes, Policies and Patients. Washingtion, DC: National Academy Press; 1991:1-312.
  4. Annual Report of the US Scientific Registry for Organ Transplantation and the Organ Procurement and Transplantation Network - 1990. Washingtion, DC: US Department of Health and Human Services: 1990.
  5. Frazier OH, Rose EA, Macmanus Q, et al. Multicenter clinical evaluation of the Heartmate 1000 IP left ventricular assist device. Ann Thorac Surg 1992; 102:578-587.
  6. McCarthy PM, Rose EA, Macmanus Q, et al. Clinical experience with the Novacor ventricular assist system. J Thorac Cardiovasc Surg. 1991; 102:578-587.
  7. Oz MC, Argenziano M, Catanese KA, et al. Bridge experience with long-term implantable left ventricular assist devices. Are they an alternative to transplantation? Circulation. 1997; 95:1844-1852.
  8. Colucci WS.  Molecular and cellular mechanisms of myocardial failure.  Am J Cardiol 1997;80:15L-25L.
  9. Ravichandran LV and Puvanakrishnan R.  In vivo labeling studies on the biosynthesis and degradation of collagen in experimental myocardial myocardial infarction. Biochem Intl 1991;24:405-414.
  10. Agocha A, Lee H-W, Eghali-Webb M.  Hypoxia regulates basal and induced DNA synthesis and collagen type I production in human cardiac fibroblasts: effects of TGF-beta, thyroid hormone, angiotensis II and basic fibroblast growth factor.  J Mol Cell Cardiol 1997;29:2233-2244H.
  11. Pfeffer JM, Pfeffer MA, Fletcher PJ, Braunwald E.  Progressive ventricular remodeling in rat with myocardial infarction.  Am J Physiol 1991; 260:H1406-14.
  12. White HD, Norris RM, Brown MA, Brandt PWT, Whitlock RML, Wild CJ.  Left ventricular end systolic volume as the major determinant of survival after recovery from myocardial infarction.  Circulation 1987; 76:44-51.
  13. Nelissen-Vrancken H, Debets J, Snoeckx L, Daemen M, Smits J. Time-related normalization of maximal coronary flow in isolated perfused hearts of rats with myocardial infarction. Circulation 1996;93:349-355.
  14. Kalkman EAJ, Bilgin YM, van Haren P, van Suylen R-J, Saxena PR, Schoemaker RG.Determinants of coronary reserve in rats subjected to coronary artery ligation or aortic banding. Cardiovasc Res 1996.
  15. Heymans S, Luutun A, Nuyens D, et al.  Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.  Nat Med 1999;5:1135-1142.
  16. Hochman JS and Choo H.  Limitation of myocardial infarct expansion by reperfusion independent of myocardial salvage. Circulation 1987;75:299-306.
  17. White HD, Cross DB, Elliot JM, et al.  Long-term prognostic importance of patency of the infarct-related coronary artery after thrombolytic therapy for myocardial infarction. Circulation 1994;89:61-67.
  18. Nidorf SM, Siu SC, Galambos G, Weyman AE, Picard MH. Benefit of late coronary reperfusion on ventricular morphology and function after myocardial infarction. J Am Coll Cardiol 1992;20:307-313.
  19. MacLellan WR and Schneider MD. Genetic dissection of cardiac growth control pathways Annu. Rev. Physiol. 2000. 62:289-320.
  20. Soonpaa MH, Field LJ. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am J Physiol  272, H220-6 (1997).
  21. Kellerman S, Moore JA, Zierhut W, Zimmer HG, Campbell J, Gerdes AM. Nuclear DNA content and nucleation patterns in rat cardiac myocytes from different models of cardiac hypertrophy. J Mol Cell Cardiol 24, 497-505 (1992).
  22. Hill MF, Singal PK. Right and left myocardial antioxidant responses during heart failure subsequent to myocardial infarction. Circulation 1997 96:2414-20.
  23. Li, Y., Jenkins, C.W. , Nichols, M.A. and Xiong, Y. (1994) Cell cycle expression and p53 regulation of the cyclin-dependent kinase inhibitor p21. Oncogene, 9, 2261-2268.
  24. Steinman, R.A. , Hoffman, B. , Iro, A. , Guillouf, C. , Liebermann, D.A. and El-Houseini, M.E. (1994) Induction of p21 (WAF1/CIP1) during differentiation. Oncogene, 9, 3389-3396.
  25. Halevy, O. , Novitch, B.G. , Spicer, D.B. , Skapek, S.X. , Rhee, J. , Hannon, G.J. , Beach, D. and Lassar, A.B. (1995) Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science, 267, 1018-1021.
  26. Andres, V. and Walsh, K. (1996) Myogenin expression, cell cycle withdrawal and phenotypic differentiation are temporally separable events that precedes cell fusion upon myogenesis. J. Cell Biol., 132, 657-666.
  27. Tsurimoto, T. PCNA Binding Proteins.  Frontiers in Bioscience, 4:849-858, 1999.
  28. Levkau B, Koyama H, Raines EW, Clurman BE, Herren B, Orth K, Roberts JM, Ross R. Cleavage of p21cip1/waf1 and p27 kip1 mediates apoptosis in endothelial cells through activation of cdk2: role of a caspase cascade. Mol Cell. 1998;1:553-563.
  29. Adachi S, et al.  Cyclin A/cdk2 activation is involved in hypoxia-induced apoptosis in cardiomyocytes Circ Res. 88:408, 2001.
  30. Anversa P, and Nadal-Ginard B. Myocyte renewal and ventricular remodelling Nature 415, 240 - 243, 2002.
  31. Kajstura J, Leri A, Finato N, di Loreto N, Beltramo CA, Anversa P.  Myocyte proliferation in end-stage cardiac failure in humans.  Proc Natl Acad Sci USA 95, 8801-8805 (1998).
  32. Beltrami AP, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med 344, 1750-7 (2001).
  33. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999, 103:697-705.
  34. Tomita S, Li R-K, Weisel RD, et al.  Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999; 100:II-247.
  35. Pittenger MF, Mackay AM, Beck SC, et al.  Multilineage potential of adult human  mesenchymal stem cells.  Science 1999, 284:143-147.
  36. Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000, 6:1282-6.
  37. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest. 2001; 108: 407-14.
  38. Klug MG, Soonpaa MH, Koh GY, Field LJ (1996) Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 98:216-224.
  39. Hescheler J, Fleischmann BK, Wartenberg M, Bloch W, Kolossov E, Ji G, Addicks K, Sauer H (1999) Establishment of ionic channels and signalling cascades in the embryonic stem cell-derived primitive endoderm and cardiovascular system. Cells Tissues Organs 165:153-164.
  40. DA Taylor, BZ Atkins, P Hungspreugs, TR Jones, MC Reedy, KA Hutcheson, DD Glower, WE. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nature Medicine 1998, 4: 929-933.
  41. L Field: Future therapy for cardiovascular disease. In Proceedings of the NHLBI Workshop Cell Transplantation: Future Therapy for Cardiovascular Disease? Columbia, MD; 1998.
  42. CE Murry, RW Wiseman, SM Schwartz, SD Hauschka: Skeletal myoblast transplantation for repair of myocardial necrosis. J Clin Invest 1996, 98: 2512-2523.
  43. BZ Atkins, MT Hueman, JM Meuchel, MJ Cottman, KA Hutcheson, DA Taylor: Myogenic cell transplantation improves in vivo regional performance in infarcted rabbit myocardium. J Heart Lung Transpl 1999, 18: 1173-1180.
  44. Suzuki K, Smolenski RT, Jayakumar J, Murtuza B, Brand NJ, Yacoub MH (2000) Heat shock treatment enhances graft cell survival in skeletal myoblast transplantation to the heart. Circulation 102:III216-221.
  45. Pouzet B, Ghostine S, Vilquin J-T, Garcin I, Scorsin M, Hagege AA, Duboc D, Schwartz K, Menasche P (2001) Is skeletal myoblast transplantation clinically relevant in the era of angiotensin-converting enzyme inhibitors? Circulation 104:I223-228.
  46. Menasche P, Hagege AA, Scorsin M, Pouzet B, Desnos M, Duboc D, Schwartz K, Vilquin J-T, Marolleau J-P (2001) Myoblast transplantation for heart failure. Lancet 347:279.
  47. Beauchamp JR, Morgan JE, Pagel CN, Partridge TA (1999) Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J Cell Biol 144:1113-1122.
  48. Smythe GM, Grounds MD (2000) Exposure to tissue culture conditions can adversely affect myoblast behaviour in vivo in whole muscle grafts: implications for myoblast transfer therapy. Cell Transplant 9:379-393.
  49. Smythe GM, Hodgetts SI, Grounds MD (2001) Problems and solutions in myoblast transfer therapy. J Cell Mol Med 5:33-47.
  50. Hodgetts SI, Beilharz MW, Scalzo T, Grounds MD (2000) Why do cultured transplanted myoblasts die in vivo? DNA quantification shows enhanced survival of donor male myoblasts in host mice depleted of CD4+ and CD8+ or NK1.1+ cells. Cell Transplant 9:489-502.
  51. Maier S, Tertilt C, Chambron N, Gerauer K, Huser N, Heidecke C-D, Pfeffer K (2001) Inhibition of natural killer cells results in acceptance of cardiac allografts in CD28-/- mice. Nature Med 7:557-562.
  52. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE (2001) Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 33:907-921.
  53. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML, Michael LH, Hirschi KK, Goodell MA (2001) Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 107:1395-1402.
  54. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001, 410:701-705.
  55. Tavian M, Coulombel L, Luton D, San Clemente H, Dieterlen-Lievre F, Peault B.  Aorta-associated CD34 hematopoietic cells in the early human embryo. Blood 1996;87:67-72.
  56. Jaffredo T, Gautier R, Eichmann A, Dieterlen-Lievre F.  Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny. Development 1998;125:4575-4583.
  57. Kennedy M, Firpo M, Choi K, Wall C, Robertson S, Kabrun N, Keller G. A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 1997;386:488-493.
  58. Choi K, Kennedy M, Kazarov A, Papadimitriou, Keller G.  A common precursor for hematopoietic and endothelial cells. Development 1998;125:725-732.
  59. Elefanty AG, Robb L, Birner R, Begley CG. Hematopoietic-specific genes are not induced during in vitro differentiation of scl-null embryonic stem cells. Blood 1997;90:1435-1447.
  60. Labastie M-C, Cortes F, Romeo P-H, Dulac C, Peault B. Molecular identity of hematopoietic precursor cells emerging in the human embryo. Blood 1998;92:3624-3635.
  61. Tsai FY, Keller G, Kuo FC, Weiss M, Chen J, Rosenblatt M, Alt FA, Orkin SH. An early hematopoietic defect in mice lacking the transcription factor GATA-2. Nature 1994;371:221-225.
  62. Ogawa M, Kizumoto M, Nishikawa S, Fujimoto T, Kodama H, Nishikawa SI. Blood 1999;93:1168-1177.
  63. Asahara, T. et al. Isolation of putative progenitor cells for endothelial angiogenesis. Science 1997; 275:964-967.
  64. Folkman, J. Therapeutic angiogenesis in ischemic limbs. Circulation 1998; 97:108-110.
  65. Takahashi, T. et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat. Med. 1999; 5:434-438.
  66. Kalka, C. et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. Sci. USA 2000; 97:3422-3427.
  67. Rafii S, Shapiro F, Rimarachin J, Nachman R, Ferris B, Weksler B, Moore AS, Asch AS.  Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 1994;84:10-19.
  68. Shi Q, Rafii S, Wu MH-D, et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92:362-367.
  69. Lin Y, Weisdorf DJ, Solovey A, Hebbel RP.  Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 2000;105:71-77.
  70. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nature Med. 2001; 7: 430-6.
  71. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 1999; 18:3964-3972.
  72. Kocher A, Schuster M, Szabolcs M, Itescu S.  Cardiomyocyte regeneration after neovascularization of ischemic myocardium by human bone marrow-derived angioblasts.  Nature Medicine 2002, in press.
  73. McEwan PE, Gray GA, Sherry L, Webb DJ, Kenyon CJ. Differential effects of angiotensin II on cardiac cell proliferation and intramyocardial perivascular fibrosis in vivo.  Circulation 1998;98:2765-2773.
  74. Kawano H, Do YS, Kawano Y, Starnes V, Barr M, Law RE, Hsueh WA.  Angiotensin II has multiple profibrotic effects in human cardiac fibroblasts. Circulation 2000;101:1130-1137.
  75. Pfeffer MA, Braunwald E, Moye LA, et al. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. The SAVE investigators. N Engl J Med 1992;327:669-677.
  76. Pitt B, Segal R, Martinez FA, et al. Randomised trial of losartan versus captopril in patients over 65 with heart failure (Evaluation of Losartan in the Elderly Study, ELITE). Lancet 1997;349:747-752.
 

 

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