Alexandra Aicher

Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI)

Background—Experimental studies suggest that transplantation of blood-derived or bone marrow–derived progenitor cells beneficially affects postinfarction remodeling. The safety and feasibility of autologous progenitor cell transplantation in patients with ischemic heart disease is unknown. Methods and Results—We randomly allocated 20 patients with reperfused acute myocardial infarction (AMI) to receive intracoronary infusion of either bone marrow–derived (n=9) or circulating blood–derived progenitor cells (n=11) into the infarct artery 4.3±1.5 days after AMI. Transplantation of progenitor cells was associated with a significant increase in global left ventricular ejection fraction from 51.6±9.6% to 60.1±8.6% (P =0.003), improved regional wall motion in the infarct zone (−1.5±0.2 to −0.5±0.7 SD/chord;P <0.001), and profoundly reduced end-systolic left ventricular volumes (56.1±20 mL to 42.2±15.1 mL;P =0.01) at 4-month follow-up. In contrast, in a nonrandomized matched reference group, left ventricular ejection fraction only slightly increased from 51±10% to 53.5±7.9%, and end-systolic volumes remained unchanged. Echocardiography revealed a profound enhancement of regional contractile function (wall motion score index 1.4±0.2 at baseline versus 1.19±0.2 at follow-up;P <0.001). At 4 months, coronary blood flow reserve was significantly (P <0.001) increased in the infarct artery. Quantitative F-18-fluorodeoxyglucose–positron emission tomography analysis revealed a significant (P <0.01) increase in myocardial viability in the infarct zone. There were no differences for any measured parameter between blood-derived or bone marrow–derived progenitor cells. No signs of an inflammatory response or malignant arrhythmias were observed. Conclusions—In patients with AMI, intracoronary infusion of autologous progenitor cells appears to be feasible and safe and may beneficially affect postinfarction remodeling processes.

Distinct Populations of Cancer Stem Cells Determine Tumor Growth and Metastatic Activity in Human Pancreatic Cancer

Pancreatic adenocarcinoma is currently the fourth leading cause for cancer-related mortality. Stem cells have been implicated in pancreatic tumor growth, but the specific role of these cancer stem cells in tumor biology, including metastasis, is still uncertain. We found that human pancreatic cancer tissue contains cancer stem cells defined by CD133 expression that are exclusively tumorigenic and highly resistant to standard chemotherapy. In the invasive front of pancreatic tumors, a distinct subpopulation of CD133(+) CXCR4(+) cancer stem cells was identified that determines the metastatic phenotype of the individual tumor. Depletion of the cancer stem cell pool for these migrating cancer stem cells virtually abrogated the metastatic phenotype of pancreatic tumors without affecting their tumorigenic potential. In conclusion, we demonstrate that a subpopulation of migrating CD133(+) CXCR4(+) cancer stem cells is essential for tumor metastasis. Strategies aimed at modulating the SDF-1/CXCR4 axis may have important clinical applications to inhibit metastasis of cancer stem cells.

Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells

Endothelial nitric oxide synthase (eNOS) is essential for neovascularization. Here we show that the impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. Mice deficient in eNOS (Nos3−/−) show reduced vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increased mortality after myelosuppression. Intravenous infusion of wild-type progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization of Nos3−/− mice in a model of hind-limb ischemia, suggesting that progenitor mobilization from the bone marrow is impaired in Nos3−/− mice. Mechanistically, matrix metalloproteinase-9 (MMP-9), which is required for stem cell mobilization, was reduced in the bone marrow of Nos3−/− mice. These findings indicate that eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells. This may contribute to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic NO bioactivity.

HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway

HMG-CoA reductase inhibitors (statins) have been developed as lipid-lowering drugs and are well established to reduce morbidity and mortality from coronary artery disease. Here we demonstrate that statins potently augment endothelial progenitor cell differentiation in mononuclear cells and CD34-positive hematopoietic stem cells isolated from peripheral blood. Moreover, treatment of mice with statins increased c-kit(+)/Sca-1(+)--positive hematopoietic stem cells in the bone marrow and further elevated the number of differentiated endothelial progenitor cells (EPCs). Statins induce EPC differentiation via the PI 3-kinase/Akt (PI3K/Akt) pathway as demonstrated by the inhibitory effect of pharmacological PI3K blockers or overexpression of a dominant negative Akt construct. Similarly, the potent angiogenic growth factor VEGF requires Akt to augment EPC numbers, suggesting an essential role for Akt in regulating hematopoietic progenitor cell differentiation. Given that statins are at least as potent as VEGF in increasing EPC differentiation, augmentation of circulating EPC might importantly contribute to the well-established beneficial effects of statins in patients with coronary artery disease.

Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease

Background—Cell therapy with bone marrow–derived stem/progenitor cells is a novel option for improving neovascularization and cardiac function in ischemic heart disease. Circulating endothelial progenitor cells in patients with coronary heart disease are impaired with respect to number and functional activity. However, whether this impairment also extends to bone marrow–derived mononuclear cells (BM-MNCs) in patients with chronic ischemic cardiomyopathy (ICMP) is unclear. Methods and Results—BM-MNCs were isolated from bone marrow aspirates in 18 patients with ICMP (ejection fraction, 38±11%) and 8 healthy control subjects (controls). The number of hematopoietic stem/progenitor cells (CD34+/CD133+), CD49d+ (VLA-4) cells, and CXCR4+ cells did not differ between the 2 groups. However, the colony-forming capacity of BM-MNCs from patients with ICMP was significantly lower compared with BM-MNCs from healthy controls (37.3±25.0 versus 113.8±70.4 granulocyte-macrophage colony-forming units; P =0.009). Likewise, the migratory response to stromal cell–derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) was significantly reduced in BM-MNCs derived from patients with ICMP compared with BM-MNCs from healthy controls (SDF-1, 46.3±26.2 versus 108.6±40.4 cells/microscopic field, P <0.001; VEGF, 34±24.2 versus 54.8±29.3 cells/microscopic field, P =0.027). To assess the in vivo relevance of these findings, we tested the functional activity of BM-MNCs to improve neovascularization in a hindlimb animal model using nude mice. Two weeks after ligation of the femoral artery and intravenous injection of 5×105 BM-MNCs, laser Doppler–derived relative limb blood flow in mice treated with BM-MNCs from patients with ICMP was significantly lower compared with mice treated with BM-MNCs from healthy controls (0.45±0.14 versus 0.68±0.15; P <0.001). The in vivo neovascularization capacity of BM-MNCs closely correlated with the in vitro assessment of SDF-1–induced migration (r =0.78; P <0.001) and colony-forming capacity (r =0.74; P <0.001). Conclusions—BM-MNCs isolated from patients with ICMP have a significantly reduced migratory and colony-forming activity in vitro and a reduced neovascularization capacity in vivo despite similar content of hematopoietic stem cells. This functional impairment of BM-MNCs from patients with ICMP may limit their therapeutic potential for clinical cell therapy.

Transdifferentiation of Blood-Derived Human Adult Endothelial Progenitor Cells Into Functionally Active Cardiomyocytes

Background—Further to promoting angiogenesis, cell therapy may be an approach for cardiac regeneration. Recent studies suggest that progenitor cells can transdifferentiate into other lineages. However, the transdifferentiation potential of endothelial progenitor cells (EPCs) is unknown. Methods and Results—EPCs were obtained from peripheral blood mononuclear cells of healthy adults or coronary artery disease (CAD) patients by cultivating with endothelial cell medium and growth factors. After 3 days, >95% of adherent cells were functionally and phenotypically EPCs. Diacetylated LDL–labeled EPCs were then cocultivated with rat cardiomyocytes for 6 days, resulting in significant increases of EPC cell length and size to a cardiomyocyte-like morphology. Biochemically, 9.94±1.39% and 5.04±1.09% of EPCs from healthy adults (n=15) or CAD patients (n=14, P <0.01 versus healthy adults), respectively, expressed &agr;-sarcomeric actinin as measured by flow cytometry. Immunocytochemistry showed that human EPCs expressed &agr;-sarcomeric actinin, cardiac troponin I (both with partial sarcomeric organization), atrial natriuretic peptide, and myocyte enhancer factor 2. Fluo 4 imaging demonstrated calcium transients synchronized with adjacent rat cardiomyocytes in transdifferentiated human EPCs. Single-cell microinjection of Lucifer yellow and calcein-AM labeling of cardiomyocytes demonstrated gap junctional communication between 51±7% of EPCs (16 hours after labeling, n=4) and cardiomyocytes. EPC transdifferentiation into cardiomyocytes was not observed with conditioned medium but in coculture with paraformaldehyde-fixed cardiomyocytes. Conclusions—EPCs from healthy volunteers and CAD patients can transdifferentiate in vitro into functionally active cardiomyocytes when cocultivated with rat cardiomyocytes. Cell-to-cell contact but not cellular fusion mediates EPC transdifferentiation. The therapeutic use of autologous EPCs may aid cardiomyocyte regeneration in patients with ischemic heart disease.

Relevance of Monocytic Features for Neovascularization Capacity of Circulating Endothelial Progenitor Cells

Background—Transplantation of ex vivo expanded circulating endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells improves the neovascularization after critical ischemia. However, the origin of the endothelial progenitor lineage and its characteristics have not yet been clearly defined. Therefore, we investigated whether the phenotype and functional capacity of EPCs to improve neovascularization depend on their monocytic origin. Methods and Results—Monocytic CD14+ cells were isolated from mononuclear cells and incubated on fibronectin-coated dishes in endothelial medium in the presence of vascular endothelial growth factor. After 4 days of cultivation, adherent cells deriving from CD14+ or CD14− mononuclear cells showed equal expression of endothelial marker proteins and capacity for clonal expansion as determined by measuring endothelial colony-forming units. In addition, transplanted EPCs (5×105 cells) deriving from CD14+ or CD14− cells were incorporated into vascular structures of nude mice after hind-limb ischemia and significantly improved neovascularization from 0.27±0.12 (no cells) to 0.66±0.12 and 0.65±0.17, respectively (P <0.001; laser Doppler-derived relative blood flow). In contrast, no functional improvement of neovascularization was detected when freshly isolated CD14+ mononuclear cells without ex vivo expansion were used (0.33±0.17). Moreover, macrophages or dendritic cells differentiated from isolated CD14+ cells were significantly less effective in improving neovascularization than EPCs cultivated from the same starting population (P <0.01). Conclusions—These data demonstrate that EPCs can be generated from nonmonocytic CD14− peripheral blood mononuclear cells and exhibit a unique functional activity to improve neovascularization after hind-limb ischemia.

Assessment of the Tissue Distribution of Transplanted Human Endothelial Progenitor Cells by Radioactive Labeling

Background—Transplantation of endothelial progenitor cells (EPCs) improves vascularization and left ventricular function after experimental myocardial ischemia. However, tissue distribution of transplanted EPCs has not yet been monitored in living animals. Therefore, we tested whether radioactive labeling allows us to detect injected EPCs. Methods and Results—Human EPCs were isolated from peripheral blood, characterized by expression of endothelial marker proteins, and radioactively labeled with [111In]indium oxine. EPCs (106) were injected in athymic nude rats 24 hours after myocardial infarction (n=8) or sham operation (n=8). Scintigraphic images were acquired after 1, 24, 48, and 96 hours after EPC injection. Animals were then killed, and specific radioactivity was measured in different tissues. At 24 to 96 hours after intravenous injection of EPCs, ≈70% of the radioactivity was localized in the spleen and liver, with only ≈1% of the radioactivity identified in the heart of sham-operated animals. After myocardial infarction, the heart-to-muscle radioactivity ratio increased significantly, from 1.02±0.19 in sham-operated animals to 2.03±0.37 after intravenous administration of EPCs. Injection of EPCs into the left ventricular cavity increased this ratio profoundly, from 2.69±1.54 in sham-operated animals to 4.70±1.55 (P <0.05) in rats with myocardial infarction. Immunostaining of cryosections from infarcted hearts confirmed that EPCs homed predominantly to the infarct border zone. Conclusions—Although only a small proportion of radiolabeled EPCs are detected in nonischemic myocardium, myocardial infarction increases homing of transplanted EPCs in vivo profoundly. Radiolabeling might eventually provide an useful tool for monitoring the fate of transplanted progenitor cells and for clinical cell therapy.

HMG-CoA Reductase Inhibitors Reduce Senescence and Increase Proliferation of Endothelial Progenitor Cells via Regulation of Cell Cycle Regulatory Genes

Abstract— Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization of ischemic tissue. Ex vivo expansion of EPCs might be useful for potential clinical cell therapy of myocardial ischemia. However, cultivation of primary cells leads to cellular aging (senescence), thereby severely limiting the proliferative capacity. Therefore, we investigated whether statins might be able to prevent senescence of EPCs. EPCs were isolated from peripheral blood and characterized. After ex vivo cultivation, EPCs became senescent as determined by acidic &bgr;-galactosidase staining. Atorvastatin or mevastatin dose-dependently inhibited the onset of EPC senescence in culture. Moreover, atorvastatin increased proliferation of EPCs as assessed by BrdU incorporation and colony-forming capacity. Whereas geranylgeranylpyrophosphate or farnesylpyrophosphate reduced the senescence inhibitory effect of atorvastatin, NO synthase inhibition, antioxidants, or Rho kinase inhibitors had no effect. To get further insights into the underlying downstream effects of statins, we measured telomerase activity and determined the expression of various cell cycle regulatory genes by using a microarray assay. Whereas telomerase activity did not change, atorvastatin modulated expression of cell cycle genes including upregulation of cyclins and downregulation of the cell cycle inhibitor p27Kip1. Taken together, statins inhibited senescence of EPCs independent of NO, reactive oxygen species, and Rho kinase, but dependent on geranylgeranylpyrophosphate. Atorvastatin-mediated prevention of EPC senescence appears to be mediated by the regulation of various cell cycle proteins. The inhibition of EPC senescence and induction of EPC proliferation by statins in vitro may importantly improve the functional activity of EPCs for potential cell therapy.

Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization

Forkhead box O (Foxo) transcription factors are emerging as critical transcriptional integrators among pathways regulating differentiation, proliferation, and survival, yet the role of the distinct Foxo family members in angiogenic activity of endothelial cells and postnatal vessel formation has not been studied. Here, we show that Foxo1 and Foxo3a are the most abundant Foxo isoforms in mature endothelial cells and that overexpression of constitutively active Foxo1 or Foxo3a, but not Foxo4, significantly inhibits endothelial cell migration and tube formation in vitro. Silencing of either Foxo1 or Foxo3a gene expression led to a profound increase in the migratory and sprout-forming capacity of endothelial cells. Gene expression profiling showed that Foxo1 and Foxo3a specifically regulate a nonredundant but overlapping set of angiogenesis- and vascular remodeling-related genes. Whereas angiopoietin 2 (Ang2) was exclusively regulated by Foxo1, eNOS, which is essential for postnatal neovascularization, was regulated by Foxo1 and Foxo3a. Consistent with these findings, constitutively active Foxo1 and Foxo3a repressed eNOS protein expression and bound to the eNOS promoter. In vivo, Foxo3a deficiency increased eNOS expression and enhanced postnatal vessel formation and maturation. Thus, our data suggest an important role for Foxo transcription factors in the regulation of vessel formation in the adult.