Young-sup Yoon

Intramyocardial Transplantation of Autologous Endothelial Progenitor Cells for Therapeutic Neovascularization of Myocardial Ischemia

Background—We investigated whether catheter-based, intramyocardial transplantation of autologous endothelial progenitor cells can enhance neovascularization in myocardial ischemia. Methods and Results—Myocardial ischemia was induced by placement of an ameroid constrictor around swine left circumflex artery. Four weeks after constrictor placement, CD31+ mononuclear cells (MNCs) were freshly isolated from the peripheral blood of each animal. After overnight incubation of CD31+ MNCs in noncoated plates, nonadhesive cells (NA/CD31+ MNCs) were harvested as the endothelial progenitor cell–enriched fraction. Nonadhesive CD31− cells (NA/CD31− MNCs) were also prepared. Autologous transplantation of 107 NA/CD31+ MNCs, 107 NA/CD31− MNCs, or PBS was performed with a NOGA mapping injection catheter to target ischemic myocardium. In a parallel study, 105 human CD34+ MNCs, 105 human CD34− MNCs, or PBS was transplanted into ischemic myocardium of nude rats 10 minutes after ligation of the left anterior descending coronary artery. In the swine study, ischemic area by NOGA mapping, Rentrop grade angiographic collateral development, and echocardiographic left ventricular ejection fraction improved significantly 4 weeks after transplantation of NA/CD31+ MNCs but not after injection of NA/CD31− MNCs or PBS. Capillary density in ischemic myocardium 4 weeks after transplantation was significantly greater in the NA/CD31+ MNC group than the control groups. In the rat study, echocardiographic left ventricular systolic function and capillary density were significantly better preserved in the CD34+ MNC group than in the control groups 4 weeks after myocardial ischemia. Conclusions—These favorable outcomes encourage future clinical trials of catheter-based, intramyocardial transplantation of autologous CD34+ MNCs in the setting of chronic myocardial ischemia.

Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction

We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis.

Unexpected Severe Calcification After Transplantation of Bone Marrow Cells in Acute Myocardial Infarction

Background— There has been a rapid increase in the number of clinical trials using unselected bone marrow (BM) cells or the mononuclear fraction of BM cells for treating ischemic heart diseases. Thus far, no significant deleterious effects or complications have been reported in any studies using BM-derived cells for treatment of various cardiac diseases. Methods and Results— Seven-week-old female Fisher-344 rats underwent surgery to induce acute myocardial infarction and were randomized into 3 groups of 16 rats, each receiving intramyocardial injection of either 7×105 DiI-labeled total BM cells (TBMCs), the same number of DiI-labeled, clonally expanded BM multipotent stem cells, or the same volume of phosphate-buffered saline in the peri-infarct area. Echocardiography 2 weeks after cell transplantation indicated intramyocardial calcification in 4 of 14 surviving rats (28.5%) in the TBMC group. Histological examination with hematoxylin and eosin staining and von Kossa staining confirmed the presence of extensive intramyocardial calcification. Alkaline phosphatase staining revealed strong positivity surrounding the calcified area suggestive of ongoing osteogenic activity. Fluorescent microscopic examination revealed that acellular calcific areas were surrounded by DiI-labeled TBMCs, suggesting the direct involvement of transplanted TBMCs in myocardial calcification. In contrast, in hearts receiving equal volumes of saline or BM multipotent stem cells delivered in the same manner, there was no evidence of calcification. Conclusions— These results demonstrate that direct transplantation of unselected BM cells into the acutely infarcted myocardium may induce significant intramyocardial calcification.

Progressive Attenuation of Myocardial Vascular Endothelial Growth Factor Expression Is a Seminal Event in Diabetic Cardiomyopathy Restoration of Microvascular Homeostasis and Recovery of Cardiac Function in Diabetic Cardiomyopathy After Replenishment of Local Vascular Endothelial Growth Factor

Background—Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis, which leads to progressive heart failure; however, the pathogenesis of DCM remains uncertain. Methods and Results—Using the streptozotocin-induced diabetic rat model, we evaluated the natural course of DCM over a period of 1 year by serial echocardiography, Western blot analysis for vascular endothelial growth factor (VEGF), endothelial progenitor cell assays, myocardial blood flow measurements, and histopathologic analysis that included terminal dUTP nick end-labeling (TUNEL), capillary and cardiomyocyte density, and fibrosis area. Downregulation of myocardial VEGF expression preceded all other features of DCM and was followed by increased apoptosis of endothelial cells, decreased numbers of circulating endothelial progenitor cells, decreased capillary density, and impaired myocardial perfusion. Apoptosis and necrosis of cardiomyocytes ensued, along with fibrosis and progressive diastolic and then systolic dysfunction. To provide further evidence of the central role of VEGF in the pathophysiology of DCM, we replenished myocardial VEGF expression using naked DNA gene therapy via direct intramyocardial injection of plasmid DNA encoding VEGF (phVEGF165). VEGF-replenished rats showed increased capillary density, decreased endothelial cell and cardiomyocyte apoptosis, and in situ differentiation of bone marrow–derived endothelial progenitor cells into endothelial cells. These anatomic findings were accompanied by significant improvements in cardiac function. Conclusions—These findings suggest that downregulation of VEGF may compromise microvascular homeostasis in the myocardium and thereby play a central role in the pathogenesis of DCM.

Role of host tissues for sustained humoral effects after endothelial progenitor cell transplantation into the ischemic heart

Noncellular differentiation effects have emerged as important mechanisms mediating therapeutic effects of stem or progenitor cell transplantation. Here, we investigated the expression patterns and sources of humoral factors and their regional and systemic biological effects after bone marrow (BM)-derived endothelial progenitor cell (EPC) transplantation into ischemic myocardium. Although most of the transplanted EPCs disappeared within a week, up-regulation of multiple humoral factors was sustained for longer than two weeks, which correlated well with the recovery of cardiac function. To determine the source of the humoral factors, we injected human EPCs into immunodeficient mice. Whereas the expression of human EPC (donor)-derived cytokines rapidly decreased to a nondetectable level within a week, up-regulation of mouse (recipient)-derived cytokines, including factors that could mobilize BM cells, was sustained. Histologically, we observed higher capillary density, a higher proliferation of myocardial cells, a lower cardiomyocyte apoptosis, and reduced infarct size. Furthermore, after EPC transplantation, BM-derived stem or progenitor cells were increased in the peripheral circulation and incorporated into the site of neovascularization and myocardial repair. These data indicate that myocardial EPC transplantation induces humoral effects, which are sustained by host tissues and play a crucial role in repairing myocardial injury.

Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling

Sonic hedgehog (Shh) is a crucial regulator of organ development during embryogenesis. We investigated whether intramyocardial gene transfer of naked DNA encoding human Shh (phShh) could promote a favorable effect on recovery from acute and chronic myocardial ischemia in adult animals, not only by promoting neovascularization, but by broader effects, consistent with the role of this morphogen in embryogenesis. After Shh gene transfer, the hedgehog pathway was upregulated in mammalian fibroblasts and cardiomyocytes. This resulted in preservation of left ventricular function in both acute and chronic myocardial ischemia by enhanced neovascularization, and reduced fibrosis and cardiac apoptosis. Shh gene transfer also enhanced the contribution of bone marrow–derived endothelial progenitor cells to myocardial neovascularization. These data suggest that Shh gene therapy may have considerable therapeutic potential in individuals with acute and chronic myocardial ischemia by triggering expression of multiple trophic factors and engendering tissue repair in the adult heart.

Functional disruption of α4 integrin mobilizes bone marrow-derived endothelial progenitors and augments ischemic neovascularization

The cell surface receptor α4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of α4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of α4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively α4 integrin–expressing cells. In vivo, a single dose of anti–α4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti–α4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti–α4 integrin ex vivo or collected from α4 integrin–deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that α4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of α4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

Myocardial regeneration with bone‐marrow‐derived stem cells

Despite significant therapeutic advances, heart failure remains the predominant cause of mortality in the Western world. Ischaemic cardiomyopathy and myocardial infarction are typified by the irreversible loss of cardiac muscle (cardiomyocytes) and vasculature composed of endothelial cells and smooth muscle cells, which are essential for maintaining cardiac integrity and function. The recent identification of adult and embryonic stem cells has triggered attempts to directly repopulate these tissues by stem cell transplantation as a novel therapeutic option. Reports describing provocative and hopeful examples of myocardial regeneration with adult bone‐marrow‐derived stem and progenitor cells have increased the enthusiasm for the use of these cells, yet many questions remain regarding their therapeutic potential and the mechanisms responsible for the observed therapeutic effects. In this review article we discuss the current preclinical and clinical advances in bone‐marrow‐derived stem or progenitor cell therapies for regeneration or repair of the ischaemic myocardium and their multiple related mechanisms involved in myocardial repair and regeneration.

Neuronal Nitric Oxide Synthase Mediates Statin-Induced Restoration of Vasa Nervorum and Reversal of Diabetic Neuropathy

Background—Peripheral neuropathy is a frequent and major complication of diabetes. Methods and Results—Severe peripheral neuropathy developed in type II diabetic mice, characterized by significant slowing of motor and sensory nerve conduction velocities. Rosuvastatin restored nerve vascularity, including vessel size, and nerve function also recovered to the levels of nondiabetic mice. Neuronal nitric oxide synthase expression in sciatic nerves was reduced in diabetic mice but was preserved by rosuvastatin. Coadministration of a nitric oxide synthase inhibitor with rosuvastatin attenuated the beneficial effects of rosuvastatin on nerve function and limited the recovery of vasa nervorum and nerve function. In vitro, rosuvastatin inhibited downregulation of neuronal nitric oxide synthase expression induced by high-glucose conditions in cultured Schwann cells. Furthermore, Akt phosphorylation in Schwann cells, downregulated by high-glucose conditions, was also restored by rosuvastatin, consistent with the change of neuronal nitric oxide synthase expression. Akt inhibition independently reduced neuronal nitric oxide synthase expression in Schwann cells in low-glucose cultures. Conclusions—These data indicate that the HMG-CoA reductase inhibitor rosuvastatin has a favorable effect on diabetic neuropathy independent of its cholesterol-lowering effect. Our data provide evidence that this effect may be mediated in part via neuronal nitric oxide synthase/nitric oxide and phosphatidylinositol 3-kinase/Akt-signaling pathways and also suggest that restoration or preservation of the microcirculation of the sciatic nerve may be involved.

Therapeutic myocardial angiogenesis with vascular endothelial growth factors

Emerging evidence has shown that administration of angiogenic growth factors, either as recombinant protein or by gene transfer, can augment tissue perfusion through neovascularization in animal models of myocardial and hindlimb ischemia. Many cytokines have angiogenic activity; one of those that have been best studied in animal models and clinical trials is vascular endothelial growth factor (VEGF). VEGF has been known to be a key regulator of physiologic and pathologic angiogenesis associated with tumor. Recently the effect of VEGF is not restricted to the direct angiogenic effect in vivo but includes mobilization of bone-marrow-derived endothelial progenitor cells and augmentation of postnatal vasculogenesis in situ. Clinical trials of therapeutic angiogenesis with VEGF in patients with end-stage coronary artery disease have shown increases in exercise time and reductions in anginal symptoms and have provided objective evidence of improved perfusion and left ventricular function. Larger scale placebo-controlled trials with recombinant protein (rhVEGF165) have been limited to intracoronary and intravenous administration and have shown favorable trends in exercise time and angina frequency. Small-scale, placebo-controlled, randomized clinical trials of gene transfer (phVEGF-2) via thoracotomy or percutaneous intramyocardial delivery demonstrated significant improvement of both subjective symptoms and objective measures of myocardial ischemia. Both therapeutic modalities appear to be safe and well tolerated. Further studies are required to determine the optimal dose, formulation, route of administration, and combinations of growth factors and the utility of adjunctive endothelial progenitor cell or other stem cell supplementation, to provide safe and effective therapeutic myocardial neovascularization. (Mol Cell Biochem 264: 63–74, 2004)