Improved heart repair upon myocardial infarction: Combination of magnetic nanoparticles and tailored magnets strongly increases engraftment of myocytes.

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (<5%) strongly hampering its efficacy. Herein we combine magnetic nanoparticle (MNP) loading of EGFP labeled embryonic cardiomyocytes (eCM) and embryonic stem cell-derived cardiomyocytes (ES-CM) with application of custom designed magnets to enhance their short and long-term engraftment. To optimize cellular MNP uptake and magnetic force within the infarct area, first numerical simulations and experiments were performed in vitro. All tested cell types could be loaded efficiently with SOMag5-MNP (200 pg/cell) without toxic side effects. Application of a 1.3 T magnet at 5 mm distance from the heart for 10 min enhanced engraftment of both eCM and ES-CM by approximately 7 fold at 2 weeks and 3.4 fold (eCM) at 8 weeks after treatment respectively and also strongly improved left ventricular function at all time points. As underlying mechanisms we found that application of the magnetic field prevented the initial dramatic loss of cells via the injection channel. In addition, grafted eCM displayed higher proliferation and lower apoptosis rates. Electron microscopy revealed better differentiation of engrafted eCM, formation of cell to cell contacts and more physiological matrix formation in magnet-treated grafts. These results were corroborated by gene expression data. Thus, combination of MNP-loaded cells and magnet-application strongly increases long-term engraftment of cells addressing a major shortcoming of cardiomyoplasty.

Overexpression of Cx43 in cells of the myocardial scar: Correction of post-infarct arrhythmias through heterotypic cell-cell coupling.

Ventricular tachycardia (VT) is the most common and potentially lethal complication following myocardial infarction (MI). Biological correction of the conduction inhomogeneity that underlies re-entry could be a major advance in infarction therapy. As minimal increases in conduction of infarcted tissue markedly influence VT susceptibility, we reasoned that enhanced propagation of the electrical signal between non-excitable cells within a resolving infarct might comprise a simple means to decrease post-infarction arrhythmia risk. We therefore tested lentivirus-mediated delivery of the gap-junction protein Connexin 43 (Cx43) into acute myocardial lesions. Cx43 was expressed in (myo)fibroblasts and CD45+ cells within the scar and provided prominent and long lasting arrhythmia protection in vivo. Optical mapping of Cx43 injected hearts revealed enhanced conduction velocity within the scar, indicating Cx43-mediated electrical coupling between myocytes and (myo)fibroblasts. Thus, Cx43 gene therapy, by direct in vivo transduction of non-cardiomyocytes, comprises a simple and clinically applicable biological therapy that markedly reduces post-infarction VT.

Glyoxalase 1-knockdown in human aortic endothelial cells - effect on the proteome and endothelial function estimates.

Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.

MicroRNA-224 is Readily Detectable in Urine of Individuals with Diabetes Mellitus and is a Potential Indicator of Beta-Cell Demise.

MicroRNA (miRNA) are a class of non-coding, 19-25 nucleotide RNA critical for network-level regulation of gene expression. miRNA serve as paracrine signaling molecules. Using an unbiased array approach, we previously identified elevated levels of miR-224 and miR-103 to be associated with a monogenic form of diabetes; HNF1A-MODY. miR-224 is a novel miRNA in the field of diabetes. We sought to explore the role of miR-224 as a potential biomarker in diabetes, and whether such diabetes-associated-miRNA can also be detected in the urine of patients. Absolute levels of miR-224 and miR-103 were determined in the urine of n = 144 individuals including carriers of a HNF1A mutation, participants with type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM) and normal controls. Expression levels were correlated with clinical and biochemical parameters. miR-224 was significantly elevated in the urine of carriers of a HNF1A mutation and participants with T1DM. miR-103 was highly expressed in urine across all diabetes cohorts when compared to controls. For both miR-224 and-103, we found a significant correlation between serum and urine levels (p < 0.01). We demonstrate that miRNA can be readily detected in the urine independent of clinical indices of renal dysfunction. We surmise that the differential expression levels of miR-224 in both HNF1A-MODY mutation carriers and T1DM may be an attempt to compensate for beta-cell demise.

Impact of GLO1 knock down on GLUT4 trafficking and glucose uptake in L6 myoblasts.

Methylglyoxal (MG), a highly reactive α-dicarbonyl metabolite of glucose degradation pathways, protein and fatty acid metabolism, plays an important role in the pathogenesis of diabetic complications. Hyperglycemia triggers enhanced production of MG and increased generation of advanced glycation endproducts (AGEs). In non-enzymatic reactions, MG reacts with arginine residues of proteins to form the AGEs argpyrimidine and hydroimidazolone. Glyoxalase 1 (GLO1), in combination with glyoxalase 2 and the co-factor glutathione constitute the glyoxalase system, which is responsible for the detoxification of MG. A GLO1 specific knock down results in accumulation of MG in targeted cells. The aim of this study was to investigate the effect of intracellularly accumulated MG on insulin signaling and on the translocation of the glucose transporter 4 (GLUT4). Therefore, L6 cells stably expressing a myc-tagged GLUT4 were examined. For the intracellular accumulation of MG, GLO1, the first enzyme of the glyoxalase pathway, was down regulated by siRNA knock down and cells were cultivated under hyperglycemic conditions (25 mM glucose) for 48 h. Here we show that GLO1 knock down augmented GLUT4 level on the cell surface of L6 myoblasts at least in part through reduction of GLUT4 internalization, resulting in increased glucose uptake. However, intracellular accumulation of MG had no effect on GLUT4 concentration or modification. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. Tiron, which is also a well-known antioxidant, had no impact on MG-induced GLUT4 translocation.