Activation of peroxisome proliferator-activated receptor (PPAR) delta by GW501516, a specific PPARdelta ligand, significantly inhibited interleukin (IL)-1beta-induced proliferation and migration of vascular smooth muscle cells (VSMCs). This effect of GW501516 was dependent on transforming growth factor-beta, and was mediated through the up-regulation of IL-1 receptor antagonist. The inhibitory effect of GW501516 on VSMC proliferation was associated with cell cycle arrest at the G1 to S phase transition, which was accompanied by the induction of p21 and p53 along with decreased cyclin-dependent kinase 4 expression. Inhibition of cell migration by GW501516 was associated with the down-regulation of matrix metalloproteinase (MMP)-2 and MMP-9 in IL-1beta-treated VSMCs. Inhibition of extracellular signal-regulated kinase significantly reduced the GW501516-mediated inhibition of IL-1beta-stimulated VSMC proliferation. These results suggest that PPARdelta plays an important role in the pathophysiology of diseases associated with the proliferation and migration of VSMCs.

Over-expression of aldose reductase (AR) has been observed in many cancer cells. To clarify the role of AR in tumor cells, we investigated the pathways mediating expression of the AR gene induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter. In A549 human lung adenocarcinoma cells, TPA elicited a dose- and time-dependent increase in AR mRNA level with an elevated enzyme activity. The TPA-induced increase in mRNA level and promoter activity of the AR gene was significantly attenuated in the presence of an inhibitor of protein kinase C, tyrosine kinase, or nuclear factor kappaB (NF-kappaB). TPA augmented the NF-kappaB-dependent gene transcription, indicating the involvement of NF-kappaB in this regulation. Accumulation of TPA-treated cells in S phase was almost completely abolished in the presence of ethyl 1-benzyl-3-hydroxy-2(5H)-oxopyrrole-4-carboxylate, an AR inhibitor. Taken together, TPA augmented the promoter activity of the AR gene via the activation of protein kinase and NF-kappaB. The inhibition of AR may assist in the chemotherapy of malignant tumors by suppressing the rapid growth of cancer cells.

Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.

The direct conversion of differentiated cells into undifferentiated or pluripotent cells would present scientific and medical benefits because of the potential for customized transplantation therapy. Although somatic cell nuclear transfer is one powerful way to fully reprogram somatic cells into a pluripotent state with the aid of oocyte or egg cytoplasm, the therapeutic applications of this approach have been hindered by technical complications as well as ethical objections. An alternative strategy for epigenetic reprogramming of differentiated cells into pluripotent status is desperately required. We have developed a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte extract to porcine fibroblast cells ex ovo. Porcine fibroblasts were permeabilized by 4 μg mL–1 of digitonin for 2 min at 4°C and then incubated in sturgeon’s oocyte extract for 5 h at 15 to 18°C followed by resealing of the cell membrane. We found that the sturgeon’s oocyte extract induced the reduction of overall levels of tri-methylation at lysine 9 of histone H3 (H3K9Me3), which might be related to preservation of DNA methylation in fully differentiated cells, of porcine fibroblast cells. However, permeabilized porcine fibroblasts after treatment with the extract were increasingly acetylated at lysine 9 on histone 3 (H3K9Ac), which might be associated with expression of pluripotency genes. In addition, the cells treated with the extract showed up-regulation of Oct3/4, Sox2, and Nanog gene expression. When somatic cell nuclear transfer embryos reconstructed by using the treated donor cells were transferred into surrogates, the pregnancy rate was slightly high. These results showed that sturgeon’s oocyte extract can reprogram porcine somatic cells into undifferentiated status. Further work needs to exploit the epigenetic reprogramming of differentiated cells into the undifferentiated state using different species oocyte extract.

In the context of multipotent stem cells, mesenchymal stem cells (MSC) derived from bone marrow have been identified as most promising cell types for the treatment of smooth muscle related injured tissues and organs. In the present study, the ability of porcine bone marrow derived MSC to differentiate in vitro into smooth muscle cells (SMC) was examined. MSC were isolated from domestic pig bone marrow by their readily adherent property to tissue culture plastic with fibroblast-like morphology. Cells were analysed for the expression of MSC specific markers by flow cytometer and mesenchymal lineage differentiation by following previously published protocols. Differences in values were analysed by one-way ANOVA using SPSS and data are presented as mean ± SD. Flow cytometry analysis of MSC showed the positive expression of markers, such as CD29 (97.33 ± 2.08%), CD44 (97.67 ± 1.15%), CD73 (62.33 ± 2.89%), CD90 (96.67 ± 2.08%) and vimentin (59.33 ± 2.52%). In contrast, the expression levels were significantly lower for CD34 (3.33 ± 1.53%), CD45 (3.67 ± 1.53%), major histocompatibility complex class II (MHC class II, 10.33 ± 2.52%) and swine leukocyte antigen-DR (SLA-DR, 9.67 ± 2.08%). The MSC were further confirmed by their ability to differentiate in vitro along the distinct lineages of adipocytes (Oil red O), osteocytes (von Kossa and Alizarin red) and chondrocytes (Alcian blue). Induction of SMC differentiation was performed with supplementation of porcine transforming growth factor-β (TGF-β) and recombinant human bone morphogenic protein 4 (BMP4) as described earlier (Wang et al. 2010 Tissue Eng. A 1201–1213) with minor modifications. Upon induction, porcine MSC acquired myoblast-like morphology with intracellular thin filaments. Immunofluorescence staining showed the presence of early and late markers of smooth muscle differentiation, such as α-smooth muscle actin (α-SMA), calponin, smooth muscle 22 α (SM22α) and smooth muscle-myosin heavy chain (SM-MHC) and their expression levels varied from 22.65% to 56.75%. Later, the expression of selected markers was demonstrated by Western blotting analysis. Consistent with this phenotypic characterisation, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (RT-qPCR) further showed the expression and a sequential up-regulation of transcripts for α-SMA, calponin, SM22α and SM-MHC. However, no expression of SMC-specific markers was observed in untreated MSC. In conclusion, these findings suggest the ability of porcine MSC from bone marrow to differentiate in vitro into SMC in the presence of growth factors. Further understanding of SMC differentiation with functional properties would be essential for employing porcine MSC as a useful model for cell-based tissue engineering and regeneration strategies. This work was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (2010-0010528) and BioGreen 21 (20070301034040), Republic of Korea.