Paracrine effects of cell transplantation: modifying ventricular remodeling in the failing heart

Structural ventricular remodeling determines the clinical progression of heart failure and has emerged as an important target for the development of novel medical and surgical therapeutic strategies. Cell transplantation is an innovative biologic therapy that may restore myocardial structure and function in failing hearts. With current forms of cell transplant therapy, true myocardial regeneration has been limited. However, cell transplantation can predictably limit maladaptive ventricular remodeling through multiple synergistic paracrine mechanisms. Some of the paracrine factors released by transplanted cells have been defined. These paracrine signals may provide beneficial effects by stimulating angiogenesis, limiting matrix disruption, and preventing apoptosis. In addition, cell transplantation may induce mobilization and homing of endogenous repair cells to injured myocardium through paracrine signals. Paracrine mediators released from transplanted cells work through multiple, diverse, and interrelated molecular pathways resulting in synergistic effects on the remodeling process. Although true myocardial regeneration remains the ultimate goal of cell therapy, the anti-remodeling abilities of cell transplantation can be harnessed to complement our contemporary surgical approaches for patients with myocardial injury at risk of congestive heart failure.     

Hyperglycemia exaggerates ischemia-reperfusion-induced cardiomyocyte injury: reversal with endothelin antagonism

OBJECTIVES: We have previously demonstrated an importance of endothelin-1 in diabetic patients undergoing bypass surgery. Recent evidence suggests that cardiomyocytes might also produce endothelin-1, which might directly impair myocyte contractility by increasing intracellular calcium levels. Because hyperglycemia is a potent stimulus of endothelin-1 production, we hypothesized that increased production, action, or both of endothelin-1 might be a mediator of direct cardiomyocyte injury in diabetes. Therefore we studied the effects of endothelin receptor blockers (BQ-123 and bosentan) on hyperglycemia-induced endothelin-1 production and cellular injury after ischemia-reperfusion. METHODS: Using a human ventricular heart cell model of simulated ischemia-reperfusion, we studied the effects of normoglycemia (5 mmol/L, 48 hours) and hyperglycemia (25 mmol/L, 48 hours) on cellular injury and endothelin-1 production. Furthermore, the effects of selective endothelin-A and mixed endothelin-A/B receptor antagonism (with BQ-123 and bosentan, respectively) were evaluated. RESULTS: Cellular injury, as assessed by means of trypan blue uptake, was higher in human ventricular heart cells subjected to hyperglycemia and simulated ischemia-reperfusion injury (P =.01); this effect was prevented with both BQ-123 and bosentan (P =.01). In addition, heart cells from the hyperglycemic group elaborated more endothelin-1 after ischemia-reperfusion (P =.02). CONCLUSIONS: Endothelin-1 production and cellular injury were greater in human ventricular heart cells subjected to hyperglycemic conditions and simulated ischemia-reperfusion. These effects are mediated by endothelin-A receptors because both BQ-123 and bosentan exerted similar degrees of protection. Endothelin receptor blockade is a novel strategy to improve the resistance of the diabetic heart to cardioplegic arrest and reperfusion.     

Stem cell therapy in the aging hearts of Fisher 344 rats: synergistic effects on myogenesis and angiogenesis

OBJECTIVE: Advanced age is a major risk factor for ventricular dysfunction and reduction of cardiac reserve. Finding novel approaches to prevent and attenuate heart dysfunction associated with advanced age is a major therapeutic challenge. The present study was designed to test whether engrafted embryonic stem cells could improve myocardial function in aging hearts. METHODS: Cultured mouse embryonic stem cells used for cell therapy were transfected with green fluorescent protein. Aging rats in the cell-treated group received intramyocardial injection of embryonic stem cells. Hemodynamic measurement, myocyte counting, and evaluation of blood flow were performed 6 weeks after cell transplantation. RESULTS: Embryonic stem cell therapy partially improved cardiac reserve, as reflected by the in vivo response to isoproterenol (INN: isoprenaline) stimulation in aging hearts 6 weeks after cell implantation. The functional benefits from engrafted embryonic stem cells were associated with increased myocyte numbers and enhanced left ventricular blood perfusion in the aging heart. The characteristic phenotype of engrafted embryonic stem cells was identified in the transplanted heart on the basis of green fluorescent protein-positive spots that were further demonstrated to differentiate into cardiac tissue with positive staining for cardiac alpha-myosin heavy chain. CONCLUSIONS: Regenerating cardiomyocytes and increasing regional blood perfusion in the aging heart after embryonic stem cell transplantation synergistically resulted in improvement of cardiac function. Embryonic stem cell transplantation might hold significant clinical potential in attenuating the progressive decrease of cardiac function associated with advanced aging.     

Restoration and regeneration of failing myocardium with cell transplantation and tissue engineering

Cell transplantation and the creation of bioengineered cardiovascular tissues are novel biologic approaches to restore and regenerate failing myocardium. These rapidly evolving therapies may complement and enhance other mechanical and surgical interventions for patients with congestive heart failure, providing cardiac surgeons with a wider range of treatments for patients at risk of congestive heart failure. Proof-of-concept studies have been performed in several experimental animal models of human cardiovascular disease, such as myocardial infarction and dilated cardiomyopathy. Although the exact mechanisms are unclear, cell transplantation restores cardiac function and limits ventricular dilatation. Clinical cell transplantation has been performed in a limited number of patients with encouraging preliminary results. In contrast, bioengineered muscle grafting is largely experimental but offers the promise of myocardial regeneration by replacing irreversibly damaged myocardium with healthy autologous tissue to facilitate more extensive ventricular remodeling surgery.     

Evolution of human cardiac myocyte dimension during prolonged mechanical support

In animal models using left ventricular assist systems over long time periods, myocardial cellular atrophy has been reported, raising concern that prolonged clinical use of such systems might lead to deterioration in left ventricular function. At the University of Pittsburgh, long-term clinical use of the Novacor (Baxter Healthcare Corp., Novacor Div., Oakland, Calif.) left ventricular support system for patients awaiting heart transplants has allowed study of the effects of long-term mechanical support on human subjects. This study determined that cardiac myocyte dimension is initially greater in patients with end-stage cardiac disease who require support rather than in patients with the same disease who do not require such support. Although myocyte dimension does decrease within a few days of the inception of support, this decrease merely brings cell size closer to the values usual in patients with chronic end-stage cardiac disease, and no further shrinkage is observed. Thus the Novacor left ventricular assist system does not appear associated with left ventricular atrophy, and its long-term use may not be detrimental to left ventricular function.     

Mast cell-derived cathepsin g: a possible role in the adverse remodeling of the failing human heart

BACKGROUND: The role of cardiac mast cells (MCs) in the progression to heart failure has recently become increasingly evident. Cathepsin g is a neutrophil- and mast cell-derived protease, which can convert angiotensin I to angiotensin II and thereby activate the TGF-beta pathway, resulting in myocyte necrosis, hypertrophy, and increased fibrosis. This study focuses on mast cell-derived cathepsin g in the human heart during heart failure and following mechanical unloading by means of heart-assist devices (LVADs). MATERIALS AND METHODS: Myocardial tissue was obtained from 10 patients with end-stage cardiomyopathy at the time of LVAD implantation (pre-LVAD) and following orthotopic heart transplantation (post-LAVD). In addition, biopsies of four normal hearts served as a control group. Paraffin-embedded sections were dual stained for cathepsin g and tryptase, a known marker for mast cells, using standard immunohistochemistry protocols. Total cathepsin g positive mast cells were counted. RESULTS: No cathepsin g positive MCs were found in normal hearts. However, we found evidence for cathepsin g in cardiac MCs in heart failure tissues (pre-LVAD). During heart failure, 46% of total MCs were cathepsin g positive as compared to after mechanical unloading, where only 11% of total MCs were cathepsin g positive (P<0.001). CONCLUSION: Heart failure causes an increase of myocardial MCs. We have provided evidence that cathepsin g positive MCs accumulate during heart failure and their total percentage decreases after ventricular unloading. This coincides with the decrease in myocyte necrosis, hypertrophy, and fibrosis. Thus, cathepsin g may play a role in the progression to heart failure by activating angiotensin II, leading to detrimental effects on the heart.     

Combined autologous cellular cardiomyoplasty using skeletal myoblasts and bone marrow cells for human ischemic cardiomyopathy with left ventricular assist system implantation: Report of a case

Myocardial regeneration therapy shows great promise as a treatment for heart failure. We recently introduced combined autologous cellular cardiomyoplasty with skeletal myoblasts and bone marrow cells as a treatment for human ischemic cardiomyopathy. We report the results of our first clinical trial of this technique, used to treat a patient with severe heart failure caused by ischemic cardiomyopathy who was being managed with a left ventricular assist system (LVAS). After combined cell transplantation, the patient showed signs of improved cardiac performance and angiogenesis, and reduced fibrosis.     

The possible use of stem cells in regenerative medicine: dream or reality?

Stem cells are one of the most fascinating areas in regenerative medicine today. They play a crucial role in the development and regeneration of human life and are defined as cells that continuously reproduce themselves while maintaining the ability to differentiate into various cell types. Stem cells are found at all developmental stages, from embryonic stem cells that differentiate into all cell types found in the human body to adult stem cells that are responsible for tissue regeneration. The general opinion postulates that clinical therapies based on the properties of stem cells may have the potential to change the treatment of degenerative diseases or important traumatic injuries in the “near” future. We here briefly review the literature in particularly for the liver, heart, kidney, cartilage, and bone regeneration.     

Current status of myocardial regeneration therapy

Myocardial regeneration therapy has emerged as an alternative therapy for heart failure and is expected to replace current conventional therapies. As a cell source, the presence of resident cardiac stem cells (RCSC) in the heart has been reported by many researchers. These RCSC show multi-potency and are considered to differentiate into myocytes. On the other hand, bone marrow stem cells have received the greatest attention as a source of cell transplantation therapy in the current era, with a larger number of clinical applications reported because of their ease and safety. Myoblasts have also emerged as a possible cell source for clinical applications. We previously found that myoblast-cell-sheet implantation improved cardiac function and ventricle thickness in a rat MI model. Furthermore, we conducted a pre-clinical large animal study using porcine MI and dog DCM models, and confirmed the effectiveness of skeletal myoblast sheets. Thereafter, we conducted clinical applications of skeletal myoblast implantation. It may eventually be possible to perform regeneration therapy as a routine therapeutic method.     

Recurrence of giant cell myocarditis in cardiac allograft.

BACKGROUND: Giant cell myocarditis causes essentially irreversible fulminant left ventricular dysfunction with associated conduction abnormalities and congestive failure. Response to immunosuppressive therapy is poor and cardiac transplantation is the only viable treatment option. The histologic hallmarks of giant cell myocarditis include a polymorphous inflammatory response with numerous multinucleated giant cells and extensive myocyte necrosis in a geographic pattern. There were 38 patients who received a cardiac transplant for giant cell myocarditis in the Giant Cell Myocarditis Registry. Among these patients, there were 9 recurrences of disease in the allograft. Concern has been expressed that recurrence of giant cell myocarditis in the allograft might be a contraindication for cardiac transplantation in the future. METHODS: In our single-center analysis we describe the clinical and histologic findings of 5 patients transplanted for giant cell myocarditis at the Cleveland Clinic. RESULTS: All but 1 of the patients were New York Heart Association (NYHA) class 4 with an average cardiac index (CI) of 1.52 liters/min x m(2). Of the 5 patients transplanted, 1 developed recurrent giant cell myocarditis. Routine right ventricular endomyocardial biopsy at 1 week exhibited severe multifocal myocardial fibrosis in addition to mild acute vascular rejection and mild grade 1A cellular rejection. Follow-up biopsy in this patient indicated grade IIIA moderate acute rejection in addition to multinucleated giant cells. Two distinct inflammatory processes were noted consisting of foci of T-cell inflammation identified by immunohistochemistry to be consistent with rejection, and a second inflammatory process with few mononuclear cells staining for macrophage or T-cell markers with eosinophils and myocyte necrosis consistent with giant cell myocarditis. Follow-up right ventricular endomyocardial biopsies (RVBXs) in this patient have subsequently demonstrated improvement in the degree of inflammatory infiltrate without vascular or significant cellular rejection. Vascular rejection was noted in 1 of the remaining 4 patients and was treated successfully with muramab-CD3 and plasmapheresis. CONCLUSIONS: Giant cell myocarditis should be expected to recur in the allograft and often does so concurrently with rejection. However, the disease in the allograft responds to therapy in a favorable manner, which differs dramatically from that in the native heart. This might be the result of detection of the disease at an earlier stage than in the native heart, or the immunosuppression milieu in the allograft. The favorable response to therapy suggests that the likelihood of recurrence of giant cell myocarditis should not be considered a barrier to transplantation.     

Gene transfection with human hepatocyte growth factor complementary DNA plasmids attenuates cardiac remodeling after acute myocardial infarction in goat hearts implanted with ventricular assist devices

BACKGROUND: Although a left ventricular assist device is often used to provide circulatory support until transplantation in severe heart failure, the mortality of long-term use of left ventricular assist devices remains high. We have shown that hepatocyte growth factor causes angiogenesis, antifibrosis, and antiapoptosis in the myocardium. Therefore, gene therapy with hepatocyte growth factor-complementary DNA plasmids may enhance the chance of "bridge to recovery." In this study, we performed gene therapy with hepatocyte growth factor in the impaired goat heart with a left ventricular assist device. METHODS: Cardiac impairment was induced in 6 adult goats (56-65 kg) by ligation of the coronary artery, and ventricular assist devices were installed. The hepatocyte growth factor group (HGF; n = 3) was administered human hepatocyte growth factor-complementary DNA plasmid (2.0 mg) in the myocardium. The control group (n = 3) was similarly administered beta-galactosidase plasmid. Four weeks after gene transfection, we attempted to wean all goats from the ventricular assist device. RESULTS: The myocardia transfected with human hepatocyte growth factor-complementary DNA contained human hepatocyte growth factor protein at levels as high as 1.0 +/- 0.3 ng/g tissue 3 days after transfection. After weaning from the ventricular assist device, the HGF group showed good hemodynamics, whereas the control group showed deterioration. The percentage of fractional shortening was significantly higher in the HGF group than the control group (HGF vs control, 37.9% +/- 1.7% vs 26.4% +/- 0.3%, respectively; P < .01). Left ventricular dilatation associated with myocyte hypertrophy and fibrotic changes was detected in the control group but not in the HGF group. Vascular density was markedly increased in the HGF group. CONCLUSIONS: These results suggest that gene therapy with human hepatocyte growth factor may enhance the chance of bridge to recovery in the impaired heart supported with a ventricular assist device.     

Update cardiac stem cell therapy

Chronic ischemic heart disease remains one of the most important causes of morbidity and mortality worldwide. Although revascularisation procedures and conventional drug therapy may delay ventricular remodelling, there is no basic therapeutic regime available to prevent or even reverse this process. Chronic coronary artery disease and heart failure impair quality of life and are associated with subsequent worsening of left ventricular function. In the recent past experimental and clinical studies have demonstrated the capacity of bone marrow stem cells in cardiac repair and regeneration of compromised heart muscle. Several clinical trials showed the safety and efficacy of autologous bone marrow stem cell transplantation in the patients with acute myocardial infarction or chronic ischemic heart disease. Today the therapeutic strategy of cell administration during cardiac surgery or coronary artery intervention is entering the clinical practice. Biological as well as methodological backgrounds, indications and clinical results of cardiac stem cell therapy for the treatment of acute myocardial infarction and chronic ischemic heart disease are reviewed.     

Time course of reverse remodeling of the left ventricle during support with a left ventricular assist device

BACKGROUND: Support with a left ventricular assist device leads to normalization of left ventricular chamber geometry, regression of myocyte hypertrophy, alterations in left ventricular collagen content, and normalized expression of genes involved with excitation-contraction coupling in patients with heart failure. The objective of this study was to investigate the time course of these processes. METHODS: Passive left ventricular pressure-volume relationships were obtained from explanted hearts of 19 patients with heart failure undergoing transplantation without left ventricular assist device support, 25 patients with heart failure supported before transplantation (duration of support ranging between 8 and 155 days), and 5 normal human hearts not suitable for transplantation. Left ventricular size was indexed by the volume at which left ventricular pressure reached 30 mm Hg. Left ventricular tissue samples were probed for sarcoplasmic endoreticular calcium adenosine triphosphatase 2a expression and processed for analysis of myocyte diameter and relative myocardial collagen content. RESULTS: The volume at which left ventricular pressure reached 30 mm Hg was not significantly different between hearts without and with assist device support for less than 40 days. However, the volume at which left ventricular pressure reached 30 mm Hg in patients with assist devices supported for more than 40 days was significantly smaller than that of the hearts without assist devices but was larger than that of normal hearts. A similar pattern was observed for myocyte diameter. Sarcoplasmic endoreticular calcium adenosine triphosphatase 2a expression increased to normal levels by about 20 days of support with an assist device. Relative collagen content was significantly increased in hearts supported for more than 40 days. CONCLUSION: Maximum structural reverse remodeling by left ventricular assist devices is complete by about 40 days. Molecular reverse remodeling of sarcoplasmic endoreticular calcium adenosine triphosphatase 2a expression is quicker, being complete by about 20 days.     

Xenogeneic embryonic stem cell-derived cardiomyocyte transplantation

PURPOSE: The purpose of this study was to investigate the survival of xenogeneic embryonic stem cell (ES cell)-derived cardiomyocytes transplanted into the normal myocardium. MATERIAL AND METHODS: Undifferentiated mouse ES cells carrying the enhanced green fluorescent protein (EGFP) were cultured in hanging drops and then plated onto dishes. These cells were identified as cardiomyocytes by the expression of cardiac-specific genes, recording of action potential, and immunostaining with anti-sarcomeric myosin antibody. Donor cells were injected into the normal myocardium, with cyclosporine administered daily. One week after the transplantation, we investigated donor cell survival by examining EGFP expression, hematoxylin and eosin staining, and immunostaining with anti-sarcomeric myosin antibody. RESULTS: In vitro donor cells derived from ES cells expressed myosin light chain-2v and alpha-myosin heavy chain genes, had action potentials of a ventricular myocyte type, and were stained by anti-sarcomeric myosin antibody. In vivo 1 week after transplantation, EGFP-expressed cells were detected in the cell transplanted area. No lymphocytic infiltration was observed around these cells. CONCLUSIONS: ES cell-derived cardiomyocytes survived in the normal myocardium after the transplantation, even in a discordant xenogeneic transplantation model. These results indicate that cell transplantation using cardiomyocytes derived from ES cells, even if xenogeneic represents an attractive strategy for treating heart disease.     

Fatal accelerated rejection with a prominent natural killer cell infiltrate in a heart transplant recipient with peripartum cardiomyopathy

Accelerated rejection is uncommon after cardiac transplantation. The mechanism is hypothesized to be mediated by cytotoxic T cells and anti-HLA antibodies resulting from a memory response to the donor allograft in sensitized patients. A role for Natural Killer (NK) cell in cellular rejection has also been suggested. We report a case of fulminant accelerated rejection in a heart transplant recipient, with a history of peripartum cardiomyopathy (PPMC). The patient had no pre-transplant donor specific antibody and flow cytometric T and B cell crossmatches were negative. Autopsy revealed left ventricular subendocardial and intramyocardial hemorrhage with diffuse lymphocytic infiltrates and myocyte damage (Grade 3R rejection). Immunohistochemistry revealed a large proportion (50–70%) of mature CD16 + NK cells with cytotoxic potential in the interstitium and the intra capillary compartments. This case highlights the need for evaluating the potential role of NK cells in accelerated rejection in heart transplant recipients.     

Cardioprotection by placenta-derived stromal cells in a murine myocardial infarction model

Background

Autologous cells for cell therapy of ischemic cardiomyopathy often display age- and disease-related functional impairment, whereas an allogenic immunotolerant cell product would allow off-the-shelf application of uncompromised donor cells. We investigated the cardiac regeneration potential of a novel, clinical-grade placenta-derived human stromal cell product (PLX-PAD).

Methods

PLX-PAD cells derived from human donor placentas and expanded in a three-dimensional bioreactor system were tested for surface marker expression, proangiogenic, anti-inflammatory, and immunomodulatory properties in vitro. In BALB/C mice, the left anterior descending artery was ligated and PLX-PAD cells (n = 10) or vehicle (n = 10) were injected in the infarct border zone. Four weeks later, heart function was analyzed by two-dimensional and M-mode echocardiography. Scar size, microvessel density, extracellular matrix composition, myocyte apoptosis, and PLX-PAD cell retention were studied by histology.

Results

In vitro, PLX-PAD cells displayed both proangiogenesis and anti-inflammatory properties, represented by the secretion of both vascular endothelial growth factor and angiopoietin-1 that was upregulated by hypoxia, as well as by the capacity to suppress T-cell proliferation and augment IL-10 secretion when co-cultured with peripheral blood mononuclear cells. Compared with control mice, PLX-PAD-treated hearts had better contractile function, smaller infarct size, greater regional left ventricular wall thickness, and less apoptosis after 4 wk. PLX-PAD stimulated both angiogenesis and arteriogenesis in the infarct border zone, and periostin expression was upregulated in PLX-PAD-treated hearts.

Conclusions

Clinical-grade PLX-PAD cells exert beneficial effects on ischemic myocardium that are associated with improved contractile function, and may be suitable for further evaluation aiming at clinical pilot trials of cardiac cell therapy.     

Gene transfection of hepatocyte growth factor attenuates reperfusion injury in the heart

Background. Hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, plays a role as organotrophic factor for regeneration of various organs. HGF has an angiogenic activity and exhibits a potent antiapoptotic activity in several types of cells. Although HGF and the c-Met/HGF receptor are expressed in the heart, the role of HGF in the heart has remained unknown.

Methods. After we analyzed changes in expression of endogenous HGF and c-Met mRNA levels in the rat left ventricle after myocardial infarction, the human HGF gene in hemagglutinating virus of Japan (HVJ)-liposome was transfected into the normal whole rat heart. Three days after transfection, the heart was subjected to global warm ischemia and subsequent reperfusion, followed by assessment of its cardiac functions.

Results. Both HGF and c-Met/HGF receptor mRNAs were expressed in adult rat heart, and c-Met/HGF receptor mRNA was upregulated in response to myocardial infarction. HGF-transfected heart showed significant increase of human HGF protein level in the heart. Cardiac functions in terms of the left ventricular developed pressure, maximum dp/dt, and pressure rate product in hearts with HGF gene transfection were significantly superior to those in control hearts. In addition, leakage of creatine phosphokinase in the coronary artery effluent in hearts with HGF gene transfection was significantly lower than that in control hearts.

Conclusions. These data indicated that both HGF and c-Met/HGF receptor mRNAs were upregulated in response to myocardial ischemic injury, and that HGF is likely to have a cytoprotective effect on cardiac tissue, presumably through the c-Met/HGF receptor.     

Identification of renal progenitor-like tubular cells that participate in the regeneration processes of the kidney

The present study was conducted to explore renal progenitor-like cells that are actively engaged in tubular regeneration after injury. For addressing this issue, the existence of label-retaining cells (LRC; slow-cycling cells) in normal rat kidneys by in vivo bromodeoxyuridine (BrdU) labeling was examined. LRC were scattering among renal epithelial tubular cells of normal rat kidneys. During the recovery after renal ischemia, LRC underwent cell division and most of them became positive for proliferating cell nuclear antigen. In contrast, proliferating cell nuclear antigen-positive but BrdU-negative tubular cells were rarely observed, suggesting that cells proliferating during tubular regeneration are essentially derived from LRC. At an early phase of tubular regeneration, descendants of LRC expressed a mesenchymal marker, vimentin, and eventually became positive for an epithelial marker, E-cadherin, after multiple cell divisions. These findings suggested that LRC function as a source of regenerating cells to replace injured cells. Collectively, it was concluded that LRC are renal progenitor-like tubular cells that provide regenerating cells, which actively proliferate and eventually differentiate into epithelial cell, during tubular regeneration. It may be possible to regenerate renal tubules in vivo through the activation of LRC.     

P-GAP-43 is enriched in horizontal cell divisions throughout rat cortical development

Asymmetric cell divisions are correlated to neurogenesis in the mammalian cortex and occur often with a horizontal orientation of cell division. However, the molecular mechanisms of spindle orientation or asymmetric cell divisions are not well understood in the developing mammalian central nervous system. Here we show a new molecular marker for horizontally dividing precursors in the mammalian telencephalon. The antibody 2G12 directed against phosphorylated serine of growth associated protein 43 (GAP-43) labels postmitotic neurons and a subset of cells in mitoses in the developing rat telencephalon. 2G12 immunoreactivity was found at a high frequency in mitotic cells dividing parallel to the ventricular surface throughout neurogenesis (embryonic day 13-17) in the cerebral cortex and ganglionic eminence. Interestingly, we detected the same predominance of 2G12 immunoreactivity in horizontally dividing cells in the subventricular zone, the second proliferative layer that has recently been involved in the generation of neurons. Moreover, 2G12 immunostaining is no longer detectable in mitotic cells of the ventricular zones at E21, the onset of gliogenesis in rat telencephalon. These data imply GAP-43 phosphorylation in the phase of neuronal commitment during M-phase and present to our knowledge the first molecular correlate to horizontally dividing precursors in mammalian neurogenesis.     

Therapeutic angiogenesis and myocardial regenerative medicine for ischemic heart disease

We have a new therapeutic modality, regenerative medicine, for patients with severe ischemic heart disease. Growth factor administration and cell transplantation are available. Therapeutic angiogenesis with bone marrow cell transplantation has been used clinically with favorable results. Basic fibroblast growth factor slow-release administration has recently started to be used clinically as another angiogenic therapy. It is more potent when combined with a donor artery and omentum (Bio-CABG). Myogenic cell transplantation is in clinical trials aimed at myocardial regeneration. However, it remains unresolved how transplanted myoblasts improve cardiac function and how we can prevent fatal arrhythmia. Many are researching cardiac stem cells and embryonic stem cells as candidates for myocardial regeneration. Recently, the paracrine effects of transplanted mesenchymal stem cells in the ischemic heart have been reported to contribute to improved cardiac function. Therefore, growth factors and cytokines may play an important role in the regeneration process induced by transplanted cells. We combined cell transplanstation with growth factor administration as well as reconstructive surgery for dilated left ventricle, which yielded excellent results. Our integrated strategy may result in the maximal benefits to patients in the future.