Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells

MicroRNAs as markers to monitor endothelin-1 signalling and potential treatment in renal disease: Carcinoma - proteinuric damage - toxicity

This review highlights new developments in miRNA as diagnostic and surveillance tools in diseases damaging the renal proximal tubule mediated by endothelin in the field of renal carcinoma, proteinuric kidney disease and tubulotoxicity. A new mechanism in the miRNA regulation of proteins leads to the binding of the miRNA directly to the DNA with premature transcriptional termination and hence the formation of truncated protein isoforms (Mxi2, Vim3). These isoforms are mediated through miRNA15a or miRNA 498, respectively. ET-1 can activate a cytoplasmic complex consisting of NF-kappa B p65, MAPK p38 alpha, and PKC alpha. Consequently, PKC alpha does not transmigrate into the nucleus, which leads to the loss of suppression of a primiRNA15a, maturation of this miRNA in the cytoplasm, tubular secretion and detectability in the urine. This mechanism has been shown in renal cell carcinoma and in proteinuric disease as a biomarker for the activation of the signalling pathway. Similarly, ET-1 induced miRNA 498 transmigrates into the nucleus to form the truncated protein Vim3, which is a biomarker for the benign renal cell tumour, oncocytoma. In tubulotoxicity, ET-1 induced miRNa133a down-regulating multiple-drug-resistant related protein-2, relevant for proteinuric and cisplatin/cyclosporine A toxicity. Current advantages and limitations of miRNAs as urinary biomarkers are discussed

Exosomes as miRNA Carriers: Formation–Function–Future

Exosomes, which are one of the smallest extracellular vesicles released from cells, have been shown to carry different nucleic acids, including microRNAs (miRNAs). miRNAs significantly regulate cell growth and metabolism by posttranscriptional inhibition of gene expression. The rapidly changing understanding of exosomes’ formation and function in delivering miRNAs from cell to cell has prompted us to review current knowledge in exosomal miRNA secretion mechanisms as well as possible therapeutic applications for personalized medicine

MicroRNAs: Small but amazing, and their association with endothelin

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the expressional regulation of genes by inhibiting gene translation. MicroRNAs are recruited and incorporated into the miRISC, ribonucleoprotein complex, targeting specific mRNAs through mechanisms specific for a miRNA sequence. Here we review the biogenesis, regulation, and monitoring of miRNAs, as well as the current evidence for potential roles of miRNAs in human diseases associated with activation of the endothelin system. These diseases include cancer, kidney disease, cardiovascular diseases, inflammatory diseases, infectious diseases, and blood diseases, that may all be aggravated by aberrant miRNA expression. In this review we will also discuss regulatory mechanisms determining production of miRNA as well as measuring or targeting miRNAs as potential novel approaches for diagnosis and treatment. Targeting miRNAs possibly will allow one to detect diseases or to interfere with the progression of diseases associated with activation of the endothelin system. (c) 2012 Elsevier Inc. All rights reserved

Magnetic nanoparticle-based amplification of microRNA detection in body fluids for early disease diagnosis

Circulating biomarkers such as microRNAs (miRNAs), short noncoding RNA strands, represent prognostic and diagnostic indicators for a variety of physiological disorders making their detection and quantification an attractive approach for minimally invasive early disease diagnosis. However, highly sensitive and selective detection methods are required given the generally low abundance of miRNAs in body fluids together with the presence of large amounts of other potentially interfering biomolecules. Although a variety of miRNA isolation and detection methods have been established in clinics, they usually require trained personnel and often constitute labor-, time- and cost-intensive approaches. During the last years, nanoparticle-based biosensors have received increasing attention due to their superior detection efficiency even in very low concentration regimes. This is based on their unique physicochemical properties in combination with their high surface area that allows for the immobilization of multiple recognition sites resulting in fast and effective recognition of analytes. Among various materials, magnetic nanoparticles have been identified as useful tools for the separation, concentration, and detection of miRNAs. Here, we review state-of-the-art technology with regard to magnetic particle-based miRNA detection from body fluids, critically discussing challenges and future perspective of such biosensors while comparing their handling, sensitivity as well as selectivity against the established miRNA isolation and detection methods

MicroRNA Function in the Profibrogenic Interplay upon Chronic Liver Disease

In chronic liver disease leading to fibrosis, hepatic stellate cells (HSC) differentiate into myofibroblasts. Myofibroblastic HSC have taken center stage during liver fibrogenesis, due to their remarkable synthesis of extracellular matrix proteins, their secretion of profibrogenic mediators and their contribution to hypertension, due to elevated contractility. MicroRNAs (miRNAs) are small, noncoding RNA molecules of 19-24 nucleotides in length. By either RNA interference or inhibition of translational initiation and elongation, each miRNA is able to inhibit the gene expression of a wide panel of targeted transcripts. Recently, it was shown that altered miRNA patterns after chronic liver disease highly affect the progression of fibrosis by their potential to target the expression of extracellular matrix proteins and the synthesis of mediators of profibrogenic pathways. Here, we underline the role of miRNAs in the interplay of the profibrogenic cell communication pathways upon myofibroblastic differentiation of hepatic stellate cells in the chronically injured liver

Endothelin-1 induced Mxi-2/Ago2 complex formation resulting in 5P536 downregulation promoting breast cancer development

Increased endothelin-1 decreases PKC alpha (PKCα), resulting in high miRNA 15a levels in kidney tumors. Breast cancer cells treated with ET-1, β-estrogen, Tamoxifen, Tamoxifen + β-estrogen and Tamoxifen + ET-1 were analysed regarding miRNA 15a expression. Significantly increased miRNA 15a levels were found after ET-1, becoming further increased in Tamoxifen + ET-1 treated cells. Our group already showed that miRNA 15a induces MAPK p38 splicing resulting in a truncated product called Mxi-2, whose function has yet to be defined in tumors. We described for the first time in ET-1 induced tumor cells that Mxi-2 builds a complex with Ago2, a miRNA binding protein, which is important for the localization of miRNAs to the 3′UTR of target genes. Furthermore, we show that Mxi-2/Ago2 is important for the interaction with the miRNA 1285 which binds to the 3′end of the tumor suppressor gene p53, being responsible for the downregulation of p53. Tissue arrays from breast cancer patients were performed, analysing Mxi-2, p53 and PKCα. Since the Mxi-2 levels increase in Tamoxifen + ET-1 treated cells, we claim that increasing ET-1 levels in Tamoxifen treated breast cancer patients are responsible for decreasing p53 levels. In summary, ET-1 decreases nuclear PKCα levels, while increasing the amount of miRNA 15a. This causes high levels of Mxi-2, necessary for complex formation with Ago2. The newly identified Mxi-2/Ago2 complex interacting with miRNA 1285 leads to increased 3′UTR p53 interaction, resulting in decreased p53 levels and subsequent tumor progression. This newly identified mechanism is a possible explanation for the development of ET-1 induced tumors

Protein kinase C alpha regulates nuclear pri-microRNA 15a release as part of endothelin signaling

Endothelin-1 induced signaling is characterized by an early induction of a nuclear factor-kappa B p65/mitogen-activated phosphokinase p38 transcription complex via its A-receptor versus a late induction via diacylglycerol, and protein kinase C. A possible interaction between these two pathways and a potential function for protein kinase C in this context has not previously been elucidated. Here we report that in Caki-1 tumor cells, protein kinase C alpha is a part of the transcription complex. With importin alpha 4 and alpha 5 as chaperones, the transcription complex transmigrates into the nucleus. Protein kinase C alpha blocks the nuclear release of primicroRNA 15a by direct binding shown by electrophoretic mobility shift assay and Duolink immune histology. The expression levels of miRNA 15a can be further manipulated by transfection of si-protein kinase C alpha, or an expression vector containing protein kinase C alpha or miRNA 15. The miRNA 15a regulation by protein kinase C alpha is detectable in different malignant human tumor cell lines (renal cell carcinoma, breast carcinoma, and melanoma). Furthermore, all three cell lines harbor both endothelin receptors (ETAR/ETBR). Specific blockage of each receptor leads to major reduction of miRNA 15a expression due to increased nuclear protein kinase C alpha translocation. We conclude that the nuclear binding of pri-microRNA 15a is a novel function of protein kinase C alpha, which plays an important role in endothelin-1 mediated signaling. Since several endothelin-sensitive, malignant tumor cell lines harbor this regulation, it could indicate a more general role in tumor biology. (C) 2011 Elsevier B.V. All rights reserved