Cardiotoxicity, which may result from intense cardiac oxidative stress and inflammation, is the main limiting factor of the anticancer therapy using doxorubicin. Because statins might exert beneficial pleiotropic cardiovascular effects, among other things, by anti-inflammatory and antioxidative mechanisms, we investigated whether or not fluvastatin pretreatment can attenuate doxorubicin-induced cardiotoxicity. Five days after a single injection of doxorubicin (20 mg/kg; i.p.), left ventricular (LV) function was measured in fluvastatintreated (DoxStatin; 100 mg/kg/day, p.o.) and saline-treated (doxorubicin) mice (n = 8 per group) by a micro conductance catheter. Untreated mice served as controls (placebo; n = 8 per group). After measurement of cardiac function, LV tissues were analyzed by molecular biological and immunohistologic methods. Injection resulted in significantly impaired LV function (LV pressure, À29%; dp/dtmax, À45%; cardiac output, À68%; P < 0.05) when compared with placebo. This was associated with a significant increase in cardiac oxidative stress, inflammation and apoptotic mechanisms, as indicated by significant increased cardiac lipid peroxidation activity, protein expression of nitrotyrosine, tumor necrosis factor A and Bax (P < 0.05). In contrast, DoxStatin mice showed improved LV function (LV pressure, +24%; dp/dtmax, +87%; cardiac output, +87%; P < 0.05) when compared with untreated doxorubicin mice. This was associated with reduced cardiac expression of nitrotyrosine, enhanced expression of the mitochondrial located antioxidative SOD 2, attenuated mitochondrial apoptotic pathways, and reduced cardiac inflammatory response. Statin pretreatment attenuates doxorubicin-induced cardiotoxicity via antioxidative and antiinflammatory effects. [Cancer Res 2009;69(2):695-9]

The tightly controlled replication of hepatocytes in liver regeneration and uncontrolled proliferation of tumor cells in hepatocellular carcinoma (HCC) are often modulated by common regulatory pathways. Several microRNAs (miRNAs) are involved in HCC progression by modulating posttranscriptional expression of multiple target genes. miR‐221, which is frequently up‐regulated in HCCs, delays fulminant liver failure in mice by inhibiting apoptosis, indicating a pleiotropic role of miR‐221 in hepatocytes. Here, we hypothesize that modulation of miR‐221 targets in primary hepatocytes enhances proliferation, providing novel clues for enhanced liver regeneration. We demonstrate that miR‐221 enhances proliferation of in vitro cultivated primary hepatocytes. Furthermore, applying two‐thirds partial hepatectomy as a surgically induced liver regeneration model we show that adeno‐associated virus‐mediated overexpression of miR‐221 in the mouse liver also accelerates hepatocyte proliferation in vivo. miR‐221 overexpression leads to rapid S‐phase entry of hepatocytes during liver regeneration. In addition to the known targets p27 and p57, we identify Aryl hydrocarbon nuclear translocator (Arnt) messenger RNA (mRNA) as a novel target of miR‐221, which contributes to the pro‐proliferative activity of miR‐221. Conclusion: miR‐221 overexpression accelerates hepatocyte proliferation. Pharmacological intervention targeting miR‐221 may thus be therapeutically beneficial in liver failure by preventing apoptosis and by inducing liver regeneration. (HEPATOLOGY 2013;)

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Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for studies on disease-related developmental processes and may serve as an autologous cell source for future treatment of many hereditary diseases. New genetic engineering tools such as zinc finger nucleases and transcription activator-like effector nuclease allow targeted correction of monogenetic disorders but are very cumbersome to establish. Aiming at studies on the knockdown of a disease-causing gene, lentiviral vector-mediated expression of short hairpin RNAs (shRNAs) is a valuable option, but it is limited by silencing of the knockdown construct upon epigenetic remodeling during differentiation. Here, we propose an approach for the expression of a therapeutic shRNA in disease-specific iPSCs using third-generation lentiviral vectors. Targeting severe α-1-antitrypsin (A1AT) deficiency, we overexpressed a human microRNA 30 (miR30)-styled shRNA directed against the PiZ variant of A1AT, which is known to cause chronic liver damage in affected patients. This knockdown cassette is traceable from clonal iPSC lines to differentiated hepatic progeny via an enhanced green fluorescence protein reporter expressed from the same RNA-polymerase II promoter. Importantly, the cytomegalovirus i/e enhancer chicken β actin (CAG) promoter-driven expression of this construct is sustained without transgene silencing during hepatic differentiation in vitro and in vivo. At low lentiviral copy numbers per genome we confirmed a functional relevant reduction (-66%) of intracellular PiZ protein in hepatic cells after differentiation of patient-specific iPSCs. In conclusion, we have demonstrated that lentiviral vector-mediated expression of shRNAs can be efficiently used to knock down and functionally evaluate disease-related genes in patient-specific iPSCs.

Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration.