Publications

Abstract Background: Duchenne muscular dystrophy (DMD) is an incurable disease, caused by the mutations in the DMD gene, encoding dystrophin, an actin-binding cytoskeletal protein. Lack of functional dystrophin results in muscle weakness, degeneration, and as an outcome cardiac and respiratory failure. As there is still no cure for affected individuals, the pharmacological compounds with the potential to treat or at least attenuate the symptoms of the disease are under constant evaluation. The pleiotropic agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, known as statins, have been suggested to exert beneficial effects in the mouse model of DMD. On the other hand, they were also reported to induce skeletal-muscle myopathy. Methods: Several methods including functional assessment of muscle function via grip strength measurement and treadmill test, enzymatic assays, histological analysis of muscle damage, gene expression evaluation, and immunofluorescence staining were conducted to study simvastatin-related alterations in mdx mice. Results: In our study, simvastatin treatment of mdx mice did not result in improved running performance; however, some beneficial effect was observed when grip strength was evaluated. Creatine kinase and lactate dehydrogenase activity, markers of muscle injury, were diminished after simvastatin delivery in mdx mice. Nevertheless, no significant changes in inflammation, fibrosis, and necrosis were noted. Interestingly, simvastatin treatment led to the decreased mRNA level of embryonic myosin heavy chain isoform, a declined percentage of centrally nucleated myofibers, and miR-1 upregulation, suggesting an alteration in the muscle regeneration. However, similarly to the changes noticed in the expression of some angiogenic factors, the obtained results are muscle-dependent, being prominent in gastrocnemius muscle but not in the diaphragm. Conclusion: In conclusion, we suggest that simvastatin has the potential to ameliorate selected aspects of DMD pathology; however, possible benefits still need to be thoroughly tested.

Abstract Skeletal muscle regeneration relies on the reciprocal interaction between many types of cells. Regenerative capacity may be altered in different disorders. In our study, we investigated whether the deletion of miR-378a (miR-378) affects muscle regeneration. We subjected 6-week-old wild-type (WT) and miR-378 knockout (miR-378 –/– ) animals to the glycerol-induced muscle injury and performed analyses in various time-points. In miR-378 –/– animals, an elevated abundance of muscle satellite cells (mSCs) on day 3 was found. Furthermore, fibro-adipogenic progenitors (FAPs) isolated from the muscle of miR-378 –/– mice exhibited enhanced adipogenic potential. At the same time, lack of miR-378 did not affect inflammation, fibrosis, adipose tissue deposition, centrally nucleated fiber count, muscle fiber size, FAP abundance, and muscle contractility at any time point analyzed. To conclude, our study revealed that miR-378 deletion influences the abundance of mSCs and the adipogenic potential of FAPs, but does not affect overall regeneration upon acute, glycerol-induced muscle injury.

A new process to form shallow trench isolation for ultra-large-scale integrated devices is presented. This technique utilizes chemical mechanical polish steps to provide a virtual planar surface at the end of processing for isolations of various size, ranging from 0.5 μm to several hundred μm. Superior uniformity has been obtained on wafers of 8 in. diam processed in a productionlike environment. Good device isolation also has been found.

Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell proliferation and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1β and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1β and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC.

<p>Expression of miR-328, miR-338-3p and miR-485-5p in SMS-CTR eRMS (SMS) and CW9019 aRMS (CW).</p>

Peroxisome proliferator-activated receptor-γ (PPARγ) agonists, which have been used as insulin sensitizers in diabetic patients, may improve functions of endothelial cells (ECs). We investigated the effect of PPARγ on angiogenic activities of murine ECs and bone marrow-derived proangiogenic cells (PACs). PACs were isolated from bone marrow of 10-12 weeks old, wild type, db/db and PPARγ heterozygous animals. Cells were cultured on fibronectin and gelatin coated dishes in EGM-2MV medium. For in vitro stimulations, rosiglitazone (10 μmol/L) or GW9662 (10 μmol/L) were added to 80% confluent cell cultures for 24 hours. Angiogenic potential of PACs and ECs was tested in vitro and in vivo in wound healing assay and hind limb ischemia model. ECs and PACs isolated from diabetic db/db mice displayed a reduced angiogenic potential in ex vivo and in vitro assays, the effect partially rescued by incubation of cells with rosiglitazone (PPARγ activator). Correction of diabetes by administration of rosiglitazone in vivo did not improve angiogenic potential of isolated PACs or ECs. In a hind limb ischemia model we demonstrated that local injection of conditioned media harvested from wild type PACs improved the blood flow restoration in db/db mice, confirming the importance of paracrine action of the bone marrow-derived cells. Transcriptome analysis showed an upregulation of prooxidative and proinflammatory pathways, and downregulation of several proangiogenic genes in db/db PACs. Interestingly, db/db PACs had also a decreased level of PPARγ and changed expression of PPARγ-regulated genes. Using normoglycemic PPARγ+/- mice we demonstrated that reduced expression of PPARγ does not influence neovascularization either in wound healing or in hind limb ischemia models. In summary, activation of PPARγ by rosiglitazone improves angiogenic potential of diabetic ECs and PACs, but decreased expression of PPARγ in diabetes does not impair angiogenesis.