Diagnostic and prognostic value of miR-1 and miR-29b on adverse ventricular remodeling after acute myocardial infarction - The SITAGRAMI-miR analysis.

BACKGROUND: MicroRNAs (miRs) have shown to exert fibrotic and anti-fibrotic effects in preclinical models of acute myocardial infarction (AMI). The aim of this study was to evaluate miR-1, miR-21, miR-29b and miR-92a as circulating biomarkers for adverse ventricular remodeling (AVR) in post-AMI patients. METHODS: Plasma levels of miR-1, miR-21, miR-29b and miR-92a were measured in 44 patients of the SITAGRAMI trial population at day 4, day 9 and 6month after AMI and in 18 matched controls (CTL). MiR expression patterns were correlated with magnetic resonance imaging (MRI) parameters for AVR (absolute change (Δ) in infarct volume (IV), left ventricular ejection fraction (LVEF) and left ventricular end-diastolic volume (LVEDV) between day 4 and 6months after AMI) and a combined cardiovascular endpoint. RESULTS: Expression of miR-1, miR-21 and miR-29b but not miR-92a was increased in AMI vs. CTL cohort showing highest miR levels at d9. However, only miR-1 and miR-29b levels significantly correlated with ΔIV and showed a trend for correlation with ΔLVEF. Only miR-29b levels at day 9 correlated with ΔLVEDV at 6-month follow-up. There was no correlation of miR levels with an adverse outcome. CONCLUSION: Mir-1 and miR-29b plasma levels post-AMI correlate with IV changes. In addition, miR-29b levels are associated with changes of LVEDV over time. These results provide insights into the role of miRs as diagnostic AVR surrogate markers. Further large scale clinical trials will be needed to evaluate the real prognostic relevance of these miRs with respect to a clinical implication in the future.

Mobilisation of haemopoietic stem cells in teriparatide-treated patients.

Parathyroid hormone (PTH) is the predominant regulator of calcium/phosphate homeostasis in the human body. Beside this classical function, preclinical and clinical studies indicated a relevant role for PTH in mobilisation of bone marrow-derived cells into peripheral blood. In addition, recombinant PTH (teriparatide) was recently approved for the treatment of severe osteoporosis. Therefore, it was the aim of the present study to investigate the dynamics of haemopoietic stem cells and corresponding in peripheral blood of 13 patients with osteoporosis during treatment with teriparatide. We were able to show that administration of teriparatide is sufficient to mobilise haemopoietic stem cells into the bloodstream accompanied by an alteration of mobilising cytokines. In conclusion, teriparatide might be a useful tool in the context of stem cell mobilisation.

DPP-4 inhibition ameliorates atherosclerosis by priming monocytes into M2 macrophages.

OBJECTIVE: Glipitins are widely used for the treatment of type 2 diabetic patients. In addition to their improvement of glycemic control, animal studies have suggested an independent anti-atherosclerotic effect of gliptins. Nevertheless, recent clinical trials regarding long-term effects of gliptin therapy on vascular events have been disappointing. This discrepancy led us to better dissect the functional role of SDF-1/CXCR4 signaling as a potential mechanism underlying gliptin action. The study should give improved understanding of the potential of gliptin therapy in the prevention and treatment of atherosclerosis. METHODS AND RESULTS: In an ApoE-/- mouse model on high cholesterol diet, long-term treatment with the DPP-4 inhibitor Sitagliptin significantly reduced atherosclerosic plaque load in the aorta. Flow cytometry analyses showed an enrichment of M2 macrophages in the aortic wall under gliptin therapy. Importantly, the number of recruited CD206+ macrophages was inversely correlated with total plaque area while no correlation was found for the overall macrophage population or M1 macrophages. Blockade of CXCR4/SDF-1 signaling by AMD3100 inhibited aortic M2 accumulation and the therapeutic effect of Sitagliptin. Correspondingly, Sitagliptin shifted the polarization profile of macrophages towards a M2-like phenotype. CONCLUSION: Sitagliptin-mediated inhibition of early atherosclerosis is based on M2-polarization during monocyte differentiation via the SDF-1/CXCR4 signaling. In contrast to earlier assumptions gliptin treatment might be especially effective in prevention of atherosclerosis.

Acute pontine infarction after percutaneous coronary intervention: a very rare but devastating complication.

A 64-year-old man suffering from an acute posterior wall myocardial infarction underwent primary percutaneous coronary intervention. After several aspiration attempts, tirofiban infusion and pre- and post-dilatation, a bare-metal stent was successfully implanted in the culprit right coronary artery. While the patient did not show any neurological symptoms before or during the procedure, he exhibited hemiplegia and loss of spontaneous speech. Additional magnetic resonance imaging showed an extensive brain stem infarction. This is the first report of a brain stem infarction as a complication of percutaneous coronary intervention.

Combined anatomical and functional imaging using coronary CT angiography and myocardial perfusion SPECT in symptomatic adults with abnormal origin of a coronary artery.

There has been a lack of standardized workup guidelines for patients with congenital abnormal origin of a coronary artery from the opposite sinus (ACAOS). We aimed to evaluate the use of cardiac hybrid imaging using multi-detector row CT (MDCT) for coronary CT angiography (Coronary CTA) and stress-rest myocardial perfusion SPECT (MPS) for comprehensive diagnosis of symptomatic adult patients with ACAOS. Seventeen symptomatic patients (12 men; 54 ± 13 years) presenting with ACAOS underwent coronary CTA and MPS. Imaging data were analyzed by conventional means, and with additional use of 3D image fusion to allocate stress induced perfusion defects (PD) to their supplying coronary arteries. An anomalous RCA arose from the left anterior sinus in eight patients, an abnormal origin from the right sinus was detected in nine patients (5 left coronary arteries, LCA and 4 LCx). Five of the 17 patients (29%) demonstrated a reversible PD in MPS. There was no correlation between the anatomical variants of ACAOS and the presence of myocardial ischemia. Image fusion enabled the allocation of reversible PD to the anomalous vessel in three patients (two cases in the RCA and the other in the LCA territory); PD in two patients were allocated to the territory of artery giving rise to the anomalies, rather than the anomalies themselves. In a small cohort of adult symptomatic patients with ACAOS anomaly there was no relation found between the specific anatomical variant and the appearance of stress induced myocardial ischemia using cardiac hybrid imaging.

Connexin 40 promoter-based enrichment of embryonic stem cell-derived cardiovascular progenitor cells.

BACKGROUND: Pluripotent embryonic stem (ES) cells that can differentiate into functional cardiomyocytes as well as vascular cells in cell culture may open the door to cardiovascular cell transplantation. However, the percentage of ES cells in embryoid bodies (EBs) which spontaneously undergo cardiovascular differentiation is low (<10%), making strategies for their specific labeling and purification indispensable. METHODS: The human connexin 40 (Cx40) promoter was isolated and cloned in the vector pEGFP. The specificity of the construct was initially assessed in Xenopus embryos injected with Cx40-EGFP plasmid DNA. Stable Cx40-EGFP ES cell clones were differentiated and fluorescent cells were enriched manually as well as via fluorescence-activated cell sorting. Characterization of these cells was performed with respect to spontaneous beating as well as via RT-PCRs and immunofluorescent stainings. RESULTS: Cx40-EGFP reporter plasmid injection led to EGFP fluorescence specifically in the abdominal aorta of frog tadpoles. After crude manual enrichment of highly Cx40-EGFP-positive EBs, the appearance of cardiac and vascular structures was increased approximately 3-fold. Immunofluorescent stainings showed EGFP expression exclusively in vascular-like structures simultaneously expressing von Willebrand factor and in formerly beating areas expressing alpha-actinin. Cx40-EGFP-expressing EBs revealed significantly higher numbers of beating cardiomyocytes and vascular-like structures. Semiquantitative RT-PCRs confirmed an enhanced cardiovascular differentiation as shown for the cardiac markers Nkx2.5 and MLC2v, as well as the endothelial marker vascular endothelial cadherin. CONCLUSIONS: Our work shows the feasibility of specific labeling and purification of cardiovascular progenitor cells from differentiating EBs based on the Cx40 promoter. We provide proof of principle that the deleted CD4 (DeltaCD4) surface marker-based method for magnetic cell sorting developed by our group will be ideally suitable for transference to this promoter.

MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling.

ES-cell-based cardiovascular repair requires an in-depth understanding of the molecular mechanisms underlying the differentiation of cardiovascular ES cells. A candidate cardiovascular-fate inducer is the bHLH transcription factor MesP1. As one of the earliest markers, it is expressed specifically in almost all cardiovascular precursors and is required for cardiac morphogenesis. Here we show that MesP1 is a key factor sufficient to induce the formation of ectopic heart tissue in vertebrates and increase cardiovasculogenesis by ES cells. Electrophysiological analysis showed all subtypes of cardiac ES-cell differentiation. MesP1 overexpression and knockdown experiments revealed a prominent function of MesP1 in a gene regulatory cascade, causing Dkk-1-mediated blockade of canonical Wnt-signalling. Independent evidence from ChIP and in vitro DNA-binding studies, expression analysis in wild-type and MesP knockout mice, and reporter assays confirm that Dkk-1 is a direct target of MesP1. Further analysis of the regulatory networks involving MesP1 will be required to preprogramme ES cells towards a cardiovascular fate for cell therapy and cardiovascular tissue engineering. This may also provide a tool to elicit cardiac transdifferentiation in native human adult stem cells.

Alternatives to heart transplantation. Symposium of the "Treatment of End-stage Heart and Lung Failure" working group on October 22, 2005 in Munich.

Heart transplantation is currently the treatment of first choice in patients with end-stage refractory heart failure. But already the demand for donor organs cannot be met, and patients face long waiting times for transplantation. In the future waiting times will become even longer as life expectancy increases and the number of heart-failure patients requiring transplantation grows. Consequently, in view of the poor prognosis of the disease in its advanced stages, alternatives to heart transplantation are increasingly gaining importance. In recent years new innovative treatment methods and techniques have been developed which have already proved clinically successful in patients with end-stage heart failure, especially as bridging measures. Some of these techniques appear suitable for long-term use and could therefore serve as an alternative to heart transplantation in some patients. Interesting new avenues of research may even lead to cardiac cell replacement therapies in the future. These approaches are currently undergoing initial clinical trials. This report presents surgical and cardiologic treatments for end-stage heart failure that have already been clinically investigated as well as techniques that are still in the preclinical stage and discusses their potential as alternatives to heart transplantation.

[Embryonic stem cells. Future perspectives]. / Embryonale Stammzellen. Zukünftige Möglichkeiten.

Embryonic stem cells (ES cells) are able to differentiate into any cell type, and therefore represent an excellent source for cellular replacement therapies in the case of widespread diseases, for example heart failure, diabetes, Parkinson's disease and spinal cord injury. A major prerequisite for their efficient and safe clinical application is the availability of pure populations for direct cell transplantation or tissue engineering as well as the immunological compatibility of the transplanted cells. The expression of human surface markers under the control of cell type specific promoters represents a promising approach for the selection of cardiomyocytes and other cell types for therapeutic applications. The first human clinical trial using ES cells will start in the United States this year.

[Stem cell therapy for chronic cardiac insufficiency--therapy of the future?]. / Stammzelltherapie bei chronischer Herzinsuffizienz: Organerneuerung nach Mass?

The incidence of chronic cardiac insufficiency is constantly increasing. However, the current therapeutic possibilities during the terminal stage are limited by a lack of donor organs. For this reason, stem cell therapy is seen as a potent therapeutic option for the future. Catheter application or cytokine-mediated mobilization of autologous adult stem cells is for acute myocardial infarction safe and potentially effective; however, for chronic cardiac insufficiency, the successes have not yet been verifiable. Hence, embryonic stem cells offer a therapeutic option that cannot be ignored: These cells are pluripotent and are, theoretically, able to continue dividing in cell culture indefinitely. Through "tissue engineering" they could generate new myocardium that could be transplanted into patients suffering from chronic cardiac insufficiency to support the pump function.

Cardiomyopathies: from genetics to the prospect of treatment.

Cardiomyopathies are defined as diseases of the myocardium associated with cardiac dysfunction ranging from lifelong symptomless forms to major health problems such as progressive heart failure, arrhythmia, thromboembolism, and sudden cardiac death. They are classified by morphological characteristics as hypertrophic (HCM), dilated (DCM), arrhythmogenic right ventricular (ARVC), and restrictive cardiomyopathy (RCM). A familial cause has been shown in 50% of patients with HCM, 35% with DCM, and 30% with ARVC. In HCM, nine genetic loci and more than 130 mutations in ten different sarcomeric genes and in the gamma 2 subunit of AMP-activated protein kinase (AMPK) have been identified, suggesting impaired force production associated with inefficient use of ATP as the crucial disease mechanism. In DCM, 16 chromosomal loci with defects of several proteins also involved in the development of skeletal myopathies have been detected. These mutated cytoskeletal and nuclear transporter proteins may alter force transmission or disrupt nuclear function, resulting in cell death. Further DCM mutations have also been identified in sarcomeric genes, which indicates that different defects of the same protein can result in either HCM or DCM. In ARVC, six genetic loci and mutations in the cardiac ryanodine receptor, which controls electromechanical coupling, and in plakoglobin and desmoglobin (molecules involved in desmosomal cell-junction integrity), have been identified. Yet, no genetic linkage has been shown in RCM. Apart from disease-causing mutations, other factors, such as environment, genetic background, and the recently identified modifier genes of the renin-angiotensin, adrenergic, and endothelin systems are likely to result in the wide variety of RCM clinical presentations. Treatment options are symptomatic and are mainly focused on treatment of heart failure and prevention of thromboembolism and sudden death. Identification of patients with high risk for major arrhythmic events is important because implantable cardioverter defibrillators can prevent sudden death. Clinical and genetic risk stratification may lead to prospective trials of primary implantation of cardioverter defibrillators in people with hereditary cardiomyopathy.

Preservation of myocardial function after adenoviral gene transfer in isolated myocardium.

Adenoviral gene transfer to the heart represents a promising model for structure-function analyses. Rabbit hearts were subjected to an ex vivo perfusion protocol that achieves gene transfer in >90% of cardiac myocytes. Contractile function of isolated myocardial preparations of these hearts was then observed for 2 days in a recently developed trabecula culture system. In sham-infected hearts, the initial developed force (F(init)) (15.6 +/- 3.7 mN/mm(2); n = 12) did not change significantly after 48 h (17.0 +/- 1.9 mN/mm(2); P = 0.46). In adenovirus-infected preparations, F(init) (14.3 +/- 1. 8 mN/mm(2); n = 21) did not significantly differ from the control (P = 0.75) and was unchanged after 48 h (15.3 +/- 2.5 mN/mm(2); P = 0. 93). After 2 days of continuous contractions, we observed homogenous and high-level expression of the reporter genes LacZ coding for beta-galactosidase and Luc coding for firefly luciferase. Luciferase activity increased more than 2,500-fold from background levels of 8. 7 x 10(3 )+/- 5.0 x 10(3) relative light units (RLU)/mg protein (from hearts transfected with promotorless adenovirus with luciferase transgene construct AdNULLLuc, n = 5) to 23.4 x 10(6)+/- 11.1 x 10(6)RLU/mg protein (from hearts tranfected with adenovirus with Rous sarcoma virus promotor and luciferase transgene construct AdRSVLuc, n = 5) in infected myocardial preparations (P < 0.005). Our results demonstrate a new ex vivo approach to achieve homogenous and high-level expression of recombinant adenoviral genes in contracting myocardium without adverse functional effects.

Transgenic myocardial overexpression of fibroblast growth factor-1 increases coronary artery density and branching.

Fibroblast growth factor (FGF)-1 plays important roles during myocardial and coronary morphogenesis. FGF-1 is also involved in the physiological response of the adult heart against ischemia, which includes cardiomyocyte protection and vascular growth. In the present study, we have generated transgenic mice with specific myocardial overexpression of the gene. Transgene expression was verified by Northern blot, and increased FGF-1 protein content was assessed by Western blot and immunoconfocal microscopy. Anatomic, histomorphological, and ultrastructural analyses revealed no major morphological or developmental abnormalities of transgenic hearts. Capillary density was unaltered, whereas the density of coronary arteries, especially arterioles, was significantly increased, as was the number of branches of the main coronary arteries. In addition, the coronary flow was significantly enhanced in transgenic mice ex vivo. These differences in the anatomic pattern of the coronary vasculature are established during the second month of postnatal life. The present findings demonstrate an important role of FGF-1 in the differentiation and growth of the coronary system and suggest that it is a key regulatory molecule of the differentiation of the arterial system.

Transgenic rat hearts expressing a human cardiac troponin T deletion reveal diastolic dysfunction and ventricular arrhythmias.

OBJECTIVE: Familial hypertrophic cardiomyopathy (FHC) due to mutations of cardiac troponin T (cTnT) is associated with a high frequency of sudden death even in the absence of cardiac hypertrophy. To investigate the causal relationship of cTnT mutations and this particular phenotype, we sought to establish a transgenic rat model for the disease. METHODS: Transgenic rats were generated expressing human wild-type cTnT or two truncated cTnT molecules (del ex16, del ex15/16), resulting from an intron 15 splice donor site mutation previously observed in FHC patients. Transgenic rat hearts were characterized by histology, immunohistochemistry and in the 'working heart'. RESULTS: Human wild-type and del ex16 cTnT were stably expressed and incorporated into the sarcomere of transgenic cardiomyocytes. Del ex16 transgenic rats revealed a lower level of expression (4-5%) than human wt cTnT animals (25-40%). In the 'working heart' model del ex16 hearts exhibited significant systolic and diastolic dysfunction without cardiac hypertrophy. In contrast, human wt cTnT hearts showed improved contractile performance and moderate myocardial hypertrophy. After 6 months of daily physical exercise one del ex16 rat died suddenly and three out of five del ex16 hearts revealed ventricular tachycardia/fibrillation. No arrhythmia was observed in human wt cTnT expressors. Myofibrillar disarray was present in del ex16 hearts after training but not in human wild-type cTnT rats or non-transgenic controls. CONCLUSION: A human cTnT deletion overexpressed in transgenic rats exerts a dominant-negative effect and mimics the phenotype of FHC with diastolic dysfunction and arrhythmias. By contrast, human cTnT wild-type animals reveal a gain of function and cardiac hypertrophy without arrhythmias.

Association of nonsense mutation of dystrophin gene with disruption of sarcoglycan complex in X-linked dilated cardiomyopathy.

BACKGROUND: In a systematic analysis of inherited forms of cardiomyopathy, we previously identified a family with X-linked dilated cardiomyopathy characterised by a mutation in the rod region of dystrophin. We have now attempted to eludicate the genetic mechanism involved in this disease, as well as the role of dystrophin-associated glycoproteins. METHODS: The affected dystrophin epitope, which lacks binding to the dys-1 antibody, was analysed by single-strand conformation polymorphism analysis, reverse-transcription PCR, and DNA sequencing. Effects on dystrophin-associated glycoproteins were studied by immunohistochemistry and western blotting. FINDINGS: A translation-termination mutation (C4148T) in exon 29 of the dystrophin gene was found in all affected family members. Alternative splicing rescued the reading frame and led to the expression of a dystrophin molecule lacking 50 aminoacids both in cardiac and skeletal muscle. Immunohistochemical analysis of the dystrophin-associated proteins revealed a reduction of beta-sarcoglycan and delta-sarcoglycan in the sarcolemma of cardiac muscle but not skeletal muscle tissue. However, western blotting revealed similar amounts of sarcoglycan subunits in both tissues. INTERPRETATION: The molecular mechanism of this subtype of X-linked cardiomyopathy may be explained by a conformational change in exon-29-deleted dystrophin, resulting in disruption of the sarcoglycan assembly in heart muscle but not skeletal muscle.

Contrasting obesity phenotypes uncovered by partial leptin receptor gene deletion in transgenic mice.

Non-insulin-dependent diabetes mellitus (type 2 diabetes) is known to be a polygenic and polyfactorial disorder. Here we describe the long-term examination of a transgenic mouse line showing the disruption of the leptin receptor (Lepr, Ob-R) gene caused by transgene insertion. The absence of the expression of the long isoform Ob-Rb uncovered a strong variation of the obesity and diabetes phenotype in the homozygous mutant mice of the outbred strain used. One part of the homozygous mice developed severe persistent early-onset obesity, whereas the other part developed cachexia after having shown initial obesity in the examination period up to 26 weeks p.p. The leptin-receptor-defective mice of this line might serve as a model for the investigation of genes modulating the development and mode of expression of diabetes.