Wolfgang-Michael Franz

Endogenous circulation of bone marrow-derived cells (BMCs) was observed in patients with dilated cardiomyopathy (DCM) who showed cardiac upregulation of Vascular Cell Adhesion Protein-1 (VCAM-1). However, the underlying pathophysiology is currently unknown. Thus, we aimed to analyze circulation, migration and G-CSF-based mobilization of BMCs in a murine model of virus-induced DCM. Mice with coxsackievirus B3 (CVB3) induced DCM and healthy controls were analyzed regarding their myocardial homing factors by PCR. To determine cardiac VCAM-1 expression ELISA and immunohistochemistry were applied. Flow cytometry was performed to analyze BMCs. Cardiac diameters and function were evaluated by echocardiography before and 4 weeks after G-CSF treatment. In murine CVB3-induced DCM an increase of BMCs in peripheral blood and a decrease of BMCs in bone marrow was observed. We found an enhanced migration of Very Late Antigen-4 (VLA-4⁺) BMCs to the diseased heart overexpressing VCAM-1 and higher numbers of CD45⁻CD34⁻Sca-1⁺ and CD45⁻CD34⁻c-kit⁺ cells. Mobilization of BMCs by G-CSF boosted migration along the VCAM-1/VLA-4 axis and reduced apoptosis of cardiomyocytes. Significant improvement of cardiac function was detected by echocardiography in G-CSF-treated mice. Blocking VCAM-1 by a neutralizing antibody reduced the G-CSF-dependent effects on stem cell migration and cardiac function. This is the first study showing that in virus-induced DCM VCAM-1/VLA-4 interaction is crucial for recruitment of circulating BMCs leading to beneficial anti-apoptotic effects resulting in improved cardiac function after G-CSF-induced mobilization.

Common cardiovascular progenitor cells are characterized and induced by expression of the transcription factor MesP1. To characterize this population we used a 3.4-kb promoter fragment previously described by our group. This served to isolate MesP1-positive cells from differentiating ES stem cells via magnetic cell sorting based on a truncated CD4 surface marker. As this proximal promoter fragment omits a distal non-cardiovasculogenic enhancer region, we were able to achieve a synchronized fraction of highly enriched cardiovascular progenitors. These led to about 90 % of cells representing the three cardiovascular lineages: cardiomyocytes, endothelial cells and smooth muscle cells as evident from protein and mRNA analyses. In addition, electrophysiological and pharmacological parameters of the cardiomyocytic fraction show that almost all correspond to the multipotent early/intermediate cardiomyocyte subtype at day 18 of differentiation. Further differentiation of these cells was not impaired as evident from strong and synchronous beating at later stages. Our work contributes to the understanding of the earliest cardiovasculogenic events and may become an important prerequisite for cell therapy, tissue engineering and pharmacological testing in the culture dish using pluripotent stem cell-derived as well as directly reprogrammed cardiovascular cell types. Likewise, these cells provide an ideal source for large-scale transcriptome and proteome analyses.

Background: We performed an initial evaluation of non-invasive ECG-gated [¹⁸ F]FDG-positron emission tomography (FDG-PET) for serial measurements of left ventricular volumes and function in murine models of dilated (DCM) and ischemic cardiomyopathy (ICM), and then tested the effect of erythropoietin (EPO) treatment on DCM mice in a preliminary FDG-PET therapy monitoring study. Methods: Mice developed DCM 8 weeks after injection with Coxsackievirus B3 (CVB3), whereas ICM was induced by ligation of the left anterior descending artery. LV volumes (EDV and ESV) and the ejection fraction (LVEF) of DCM, ICM and healthy control mice were measured by FDG-PET and compared with reference standard results obtained with 1.5 T magnetic resonance imaging (MRI). In the subsequent monitoring study, LVEF of DCM mice was evaluated by FDG-PET at baseline, and after 4 weeks of treatment, with EPO or saline. Results: LV volumes and the LVEF as measured by FDG-PET correlated significantly with the MRI results. These correlations were higher in healthy and DCM mice than in ICM mice, in which LVEF measurements were somewhat compromised by absence of FDG uptake in the area of infarction. LV volumes (EDV and ESV) were systematically underestimated by FDG-PET, with net bias such that LVEF measurements in both models of heart disease exceeded by 15% to 20% results obtained by MRI. In our subsequent monitoring study of DCM mice, we found a significant decrease of LVEF in the EPO group, but not in the saline-treated mice. Moreover, LVEF in the EPO and saline mice significantly correlated with histological scores of fibrosis. Conclusions: LVEF estimated by ECG-gated FDG-PET significantly correlated with the reference standard MRI, most notably in healthy mice and mice with DCM. FDG-PET served for longitudinal monitoring of effects of EPO treatment in DCM mice.

Aims Parathyroid hormone (PTH) administration after myocardial infarction (MI) is known to attenuate ischaemic cardiomyopathy. This effect mainly resulted from an increase in mobilization and homing of CD34+/CD45+ cells into the ischaemic myocardium. PTH-related stem cell mobilization was shown to be related to endogenous granulocyte-colony stimulating factor (G-CSF) release. The aim of our study is to determine the role of G-CSF on the cardioprotective effects of PTH. Methods and results G-CSF +/+ (C57BL/6) and G-CSF −/− mice were treated with PTH for 6 days after inducing a MI. The myocardial homing factor stromal cell-derived factor-1 (SDF-1) was analysed on day 2 with enzyme-linked immunosorbent assay. Stem cell populations in peripheral blood and hearts were examined by FACS on days 6 and 2, respectively. Cardiac function and immunohistochemistry were investigated on day 6 and day 30. PTH treatment resulted in a significant increase in CD45+/CD34+ cells in peripheral blood in G-CSF +/+ but not in G-CSF −/− mice. However, a significant increase in SDF-1 and enhanced migration of CD45+/CD34+ cells into the ischaemic myocardium was revealed after PTH administration in both G-CSF +/+ and G-CSF −/− mice. Enhanced stem cell homing was associated with improved cardiac function and post-MI survival after PTH treatment. Furthermore, infarct size, wall thickness, and neovascularization showed a significant improvement in both groups 30 days after MI. Conclusion The cardioprotective effects of PTH were shown to be independent of endogenous G-CSF release and therefore from stem cell mobilization. This puts more emphasis on the role of stem cell homing into ischaemic myocardium.

Background: Left coronary artery arising from the right sinus of Valsalva is an uncommon congenital coro-nary anomaly that seems to be associated with sudden death in young patients. Case presentation: We report a case of cardiac arrest in a 59-year-old patient after sexual intercourse and Silde-nafil ingestion. A coronary arteriography and an angiographic computed tomography scan subsequently revealed a LCA origin from the right aortic sinus along with an intramural course of the left main stem. In addition a distal stenosis of the right coronary artery was detected. After successful resuscitation without neurological deficits coronary artery bypass surgery was performed. Conclusion: To our knowledge, this is the first report demonstrating sudden cardiac arrest associated with Sildenafil ingestion in a patient with this type of coronary anomaly. The question arises, whether a cardiac screening is necessary before a Sildenafil therapy is initiated.

Our recent work demonstrated that common cardiovascular progenitor cells are characterized and induced by the expression of the transcription factor mesoderm posterior1 (MesP1) in vertebrate embryos and murine embryonic stem cells. As the proliferative potential of stem cell-derived cardiomyocytes is limited, it is crucial to understand how MesP1 expression is mediated in order to achieve reasonable and reliable yields for novel stem cell-based therapeutic options. As potential upstream regulators of MesP1, we therefore analysed Eomes and Brachyury(T), which had been controversially discussed as being crucial for cardiovasculogenic lineage formation. Wild-type and transgenic murine embryonic stem cell lines, mRNA analyses, embryoid body formation, and cell sorting revealed that the MesP1 positive population emerges from the Brachyury(T) positive fraction. In situ hybridizations using wild-type mouse embryos confirmed that Brachyury(T) colocalises with MesP1 in vivo. Likewise, shRNA-based loss of Brachyury(T) causes a dramatic decrease in MesP1 expression accompanied by reduced cardiac markers in differentiating embryonic stem cells, which is reflected in vivo via in situ hybridizations using Brachyury(T) knock-out embryos where MesP1 mRNA is greatly abolished. We finally defined a 3.4 kb proximal MesP1-promoter fragment which is directly bound and activated by Brachyury(T) via a T responsive element as shown via bandshift, chromatin immuneprecipitation, and reporter assays. Our work contributes to the understanding of the earliest cardiovasculogenic events and may become an important prerequisite for cell therapy, tissue engineering, and pharmacological testing in the culture dish using pluripotent stem cell-derived as well as directly reprogrammed cardiovascular cell types.

Ischemic heart diseases are the leading cause of death in the Western world. With increasing numbers of patients surviving their acute myocardial infarction owing to effective heart catheter techniques and intensive care treatment, congestive heart failure has become an increasing health concern. With therapeutic options for the prevention and treatment of ischemic heart disease being limited at present, huge efforts have been made in the field of stem cell research to try to establish new approaches for myocardial tissue regeneration. Owing to their pronounced differentiation potential, pluripotent stem cells seem to represent the most promising cell source for future engineering of myocardial replacement tissue. However, several crucial hurdles regarding cell yield and purity of the cultured cardiovascular progenitor cells have still not been overcome to facilitate a clinical application today. By contrast, plenty of adult stem and progenitor cells have already been well characterized and investigated in human disease. However, all of these heterogeneous cell lines primarily seem to work in a paracrine manner on ischemic myocardial tissue, rather than transdifferentiating into contractile cardiomyocytes. This article will focus on the production, application and present limitations of stem cells potentially applicable for myocardial repair.

Parathyroid hormone (PTH) has been shown to promote stem cell mobilization into peripheral blood. Moreover, PTH treatment after myocardial infarction (MI) improved survival and myocardial function associated with enhanced homing of bone marrow-derived stem cells (BMCs). To unravel the molecular mechanisms of PTH-mediated stem cell trafficking, we analysed wild-type (wt) and green fluorescent protein (GFP)-transgenic mice after MI with respect to the pivotal stromal cell-derived factor-1 (SDF-1)/chemokine receptor type 4 (CXCR4) axis. WT and GFP-transgenic mice (C57BL/6J) were infarcted by coronary artery ligation and PTH (80 μg/kg/day) was injected for 6 days afterwards. Number of BMCs was analysed by flow cytometry. SDF-1 protein levels and activity of dipeptidyl peptidase-IV (DPP-IV) were investigated by ELISA and activity assay. Functional analyses were performed at day 30 after MI. PTH-treated animals revealed an enhanced homing of CXCR4⁺ BMCs associated with an increased protein level of the corresponding homing factor SDF-1 in the ischaemic heart. In vitro and in vivo, PTH inhibited the activity of DPP-IV, which cleaves and inactivates SDF-1. Functionally, PTH significantly improved myocardial function after MI. Both stem cell homing as well as functional recovery were reversed by the CXCR4 antagonist AMD3100. In summary, PTH is a DPP-IV inhibitor leading to an increased cardiac SDF-1 level, which enhances recruitment of CXCR4⁺ BMCs into the ischaemic heart associated with attenuated ischaemic cardiomyopathy. Since PTH is already clinically used our findings may have direct impact on the initiation of studies in patients with ischaemic disorders.