Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro. These sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. We now demonstrate the critical role of an EWS/FLI-bound GGAA-microsatellite in regulation of the gene as well as for Ewing sarcoma proliferation and anchorage-independent growth. Clinically, genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal "sweet-spot" GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this remains unknown. Our biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion), demonstrate a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the microsatellite was increased to the sweet-spot length. In contrast, a fully functional EWS/FLI mutant (Mut9, which retains approximately half of the EWS portion of the fusion) showed low affinity for smaller GGAA-microsatellites but instead significantly increased its affinity at sweet-spot microsatellite lengths. Single-gene ChIP and genome-wide ChIP-sequencing (ChIP-seq) and RNA-seq studies extended these findings to the in vivo setting. Together, these data demonstrate the critical requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unexpected role for the EWS portion of the EWS/FLI fusion in binding to sweet-spot GGAA-microsatellites.
Background Surgical management of long segment tracheal disease is limited by a paucity of donor tissue and poor performance of synthetic materials. A potential solution is the development of a tissue-engineered tracheal graft (TETG), which promises an autologous airway conduit with growth capacity. Methods We created a TETG by vacuum seeding bone marrow-derived mononuclear cells (BM-MNCs) on a polymeric nanofiber scaffold. First, we evaluated the role of scaffold porosity on cell seeding efficiency in vitro. We then determined the effect of cell seeding on graft performance in vivo using an ovine model. Results Seeding efficiency of normal porosity (NP) grafts was significantly increased when compared to high porosity (HP) grafts (NP: 360.3 ± 69.19 ×103 cells/mm2; HP: 133.7 ± 22.73 ×103 cells/mm2; p<0.004). Lambs received unseeded (n=2) or seeded (n=3) NP scaffolds as tracheal interposition grafts for 6 weeks. Three animals were terminated early due to respiratory complications (n=2 unseeded, n=1 seeded). Seeded TETG explants demonstrated wound healing, epithelial migration, and delayed stenosis when compared to their unseeded counterparts. Conclusion Vacuum seeding BM-MNCs on nanofiber scaffolds for immediate implantation as tracheal interposition grafts is a viable approach to generate TETGs, but further preclinical research is warranted before advocating this technology for clinical application.
Stenosis is a critical problem in the long-term efficacy of tissue-engineered vascular grafts (TEVGs). We previously showed that host monocyte infiltration and activation within the graft drives stenosis and that TGF-b receptor 1 (TGF-bR1) inhibition can prevent it, but the latter effect was attributed primarily to inhibition of mesenchymal cell expansion. In this study, we assessed the effects of TGF-bR1 inhibition on the host monocytes. Biodegradable TEVGs were implanted as inferior vena cava interposition conduits in 2 groups of C57BL/6 mice (n = 25/group): unseeded grafts and unseeded grafts with TGF-bR1 inhibitor systemic treatment for the first 2 wk. The TGF-bR1 inhibitor treatment effectively improved TEVG patency at 6 mo compared to the untreated control group (91.7 vs. 48%, P < 0.001), which is associated with a reduction in classic activation of mononuclear phagocytes. Consistent with these findings, the addition of rTGF-b to LPS/IFN-g-stimulated monocytes enhanced secretion of inflammatory cytokines TNF-a, IL-12, and IL-6; this effect was blocked by TGF-bR1 inhibition (P < 0.0001). These findings suggest that the TGF-b signaling pathway contributes to TEVG stenosis by inducing classic activation of host monocytes. Furthermore, blocking monocyte activation by TGF-bR1 inhibition provides a viable strategy for preventing TEVG stenosis while maintaining neotissue formation.-Lee, Y.-U., de Dios Ruiz-Rosado, J., Mahler, N., Best, C. A., Tara, S., Yi, T., Shoji, T., Sugiura, T., Lee, A. Y., Robledo-Avila, F., Hibino, N., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. TGF-b receptor 1 inhibition prevents stenosis of tissue-engineered vascular grafts by reducing host mononuclear phagocyte activation. FASEB J. 30, 2627FASEB J. 30, -2636FASEB J. 30, (2016. www.fasebj.orgPrevious data from our laboratory demonstrated that bone marrow-derived mononuclear cell (BM-MNC) seeding of tissue-engineered vascular grafts (TEVGs) promoted the process of neovessel formation (1). In contrast to our original expectation that the seeded cells differentiated into mature vascular cells, we found that the seeded BMMNCs disappeared within a few days of implantation. Instead, the seeded cells exerted their effect by controlling recruitment of host mononuclear phagocytes in early time point, which recruit smooth muscle cells and endothelial cells (;3 wk) (1). While BM-MNC seeding also significantly improved patency of the TEVGs, both in a clinical study and in mouse models, the occurrence of graft stenosis remained a major complication in 25% of TEVG recipients (2, 3). Our mouse models revealed that the formation of TEVG stenosis is also driven by paracrine functions of infiltrating host mononuclear phagocytes. A significant proportion of cells that form stenotic lesions in the mouse model appear to arise from the endothelialmesenchymal transition (Endo-MT) (1,3,4). Because TGFb signaling is the key to multiple vascular disorders, including Endo-MT (5-8), we investigated if inhibition of this cytokine coul...
Aim We investigated the effect of cell seeding dose and incubation time on tissue-engineered vascular graft (TEVG) patency. Materials & methods Various doses of bone marrow-derived mononuclear cells (BM-MNCs) were seeded onto TEVGs, incubated for 0 or 12 h, and implanted in C57BL/6 mice. Different doses of human BM-MNCs were seeded onto TEVGs and measured for cell attachment. Results The incubation time showed no significant effect on TEVG patency. However, TEVG patency was significantly increased in a dose-dependent manner. In the human graft, more bone marrow used for seeding resulted in increased cell attachment in a dose-dependent manner. Conclusion Increasing the BM-MNC dose and reducing incubation time is a viable strategy for improving the performance and utility of the graft.
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