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Coronary heart disease and the resulting myocardial infarction include one of the leading causes of death worldwide. Despite the progress made in the field of diagnosis and therapy, many patients suffer from heart failure after acute myocardial infarction (AMI) as a result of the restructuring processes. Until today, no adequate treatment option has been developed to regenerate the heart muscle and revitalize already lost myocardial tissue. As has been shown in several studies, an AMI leads to a physiological mobilization of endothelial progenitor cells (EPC) from the bone marrow into the peripheral blood. However, these intrinsic mechanisms are insufficient to bring about a full recovery. Therefore, one of the most promising approaches to support endogenous regeneration is the establishment of an effective neovascularization via recruitment of EPC into ischemic heart tissue. In this work various drugs and drug combinations have been applied, which have the potential to mobilize stem and progenitor cells from the bone marrow into the peripheral blood. For this reason the potential beneficial effects of mobilized hematopoietic stem and progenitor cells (HSPC) and EPC on the cardiac regeneration in the murine myocardial infarction model were investigated.
AMI was induced in 177 FVB/NJ mice by ligation of the left coronary artery. The animals were randomized into six treatment groups and the active G-CSF (granulocyte-colony stimulating factor), VEGF (vascular endothelial growth factor), AMD3100 or the combination of G-CSF/AMD3100 or VEGF/AMD3100 was administered subcutaneously after surgery. The application of G-CSF and VEGF started 1 h post OP (day 1) and was continued on the two following days (day 2 and 3). The active ingredient AMD3100 was injected on day 4. The control group received injections of saline instead of the active ingredients. Blood samples for further examination from the tail vein were collected on days 0, 7, 14 and 28. HSPC and EPC were isolated from the peripheral blood and progenitor cell numbers were evaluated by fluorescence-activated cell sorting (FACS) analysis. After 28 days, hemodynamic parameters were recorded to assess the ventricular function by means of catheter examinations. After sacrificing the mice and removal of the heart, histological studies to determine the capillary density in the cardiac tissue were performed. Furthermore infarct size and left ventricular fibrosis were measured.
The analysis of peripheral blood in all groups showed a rise of mobilized HPSC and EPC after induced heart attack. In Flow Cytometry we could demonstrate that the group that received the combined therapy with VEGF and AMD3100 mobilized most effective Flk-1+/Sca-1+ EPC into the peripheral blood. Immunofluorescence staining of CD31 EPC marker in the infarcted left ventricle demonstrated significantly enhanced capillary density in the heart tissues of VEGF/AMD3100 treated mice. The improved neovascularization consecutively led to a reduction of the infarct tissue and a well-preserved pumping function after induced myocardial infarction, which manifested itself in an increased survival rate. In contrast to this no improvement of left ventricular pump function could be observed in the mice treated with AMD3100 respectively VEGF alone, as well as in the mice treated with GCSF or G-CSF/AMD3100. No beneficial effects on the myocardial regeneration were observed in regard to the infarct size, vascularization of the ischemic myocardium and the survival rate in these groups of active ingredients.
The results indicate that the combined therapy with VEGF/AMD3100 not only promotes the endogenous mobilization of the EPC, but that the recruited cells also contribute to an improved myocardial neovascularization with positive affects on heart function after AMI. This effect was not observed in the mice who received singular active substances or the combined treatment with G-CSF and AMD3100. So for example the sole administration of VEGF led to a formation of abnormally large vascular structures. Adverse effects in terms of the myocardial regeneration were also observed in the administration of G-CSF or the combination of G-CSF and AMD3100 after AMI, like for example the increased occurrence of pericardial inflammation. In conclusion our study demonstrates that co-administration of VEGF and AMD3100 is more effective than each drug alone or treatment with G-CSF respectively G-CSF/AMD3100 with regard to EPC mobilization, myocardium vascularization, preservation of heart function after
AMI and preventing cardiac death. The use of this new combinatorial therapy that does not require ex vivo cell purification and intracoronary or intramyocardial injection of stem cells may become a therapeutic alternative for future clinical trials for cardiac repair after AMI.