Department of Cariology, Restorative Sciences and Endodontics, University of Michigan School of Dentistry, Ann Arbor, Michigan; Department of Restorative Dentistry, Institute of Science and Technology, São Paulo State University, São José dos Campos, São Paulo, Brazil.
Department of Cariology, Restorative Sciences and Endodontics, University of Michigan School of Dentistry, Ann Arbor, Michigan.
J Endod. 2017 Sep;43(9S):S25-S30. doi: 10.1016/j.joen.2017.06.006. Epub 2017 Aug 1.
The aim of this study was to evaluate the effects of Wnt signaling through lipoprotein receptor-related protein 6 (LRP6) and Frizzled6 on the endothelial differentiation of dental pulp stem cells (DPSCs). DPSCs were stably transduced with enhanced green fluorescent protein (EGFP)-tagged lentiviral vectors (short hairpin RNA-LRP6, short hairpin RNA-Frizzled6, or empty vector controls). We evaluated the effects of LRP6 and Frizzled6 on expression of endothelial markers and on capillary tube formation mediated by DPSCs induced with recombinant human Wnt1 (rhWnt1) and/or recombinant human vascular endothelial growth factor (rhVEGF). In vivo, tooth slices/scaffolds were seeded with LRP6-silenced, Frizzled6-silenced, or vector control DPSC cells and transplanted into immunodeficient mice. The density of blood vessels generated by DPSCs differentiated into vascular endothelial cells was analyzed by immunohistochemistry for EGFP. The rhWnt1 and rhVEGF induced expression of active β-catenin in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. Furthermore, VEGF and interleukin-8 were downregulated in LRP6-silenced DPSCs, but not in control DPSCs or in Frizzled6-silenced DPSCs (P < .05). Likewise, rhWnt1 and rhVEGF induced expression of the endothelial marker VEGF receptor-2 in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. These data correlated with a trend for lower density of capillary sprouts generated by LRP6-silenced DPSCs when compared with control DPSCs in Matrigel. In vivo, tooth slice/scaffolds seeded with DPSC-short hairpinRNA-LRP6 cells showed lower density of human blood vessels (ie, EGFP-positive blood vessels), when compared with tooth slice/scaffolds seeded with vector control cells (P < .05). Collectively, these data demonstrated that LRP6 signaling is necessary for the vasculogenic differentiation of human DPSCs.
本研究旨在评估 Wnt 信号通过脂蛋白受体相关蛋白 6（LRP6）和卷曲蛋白 6（Frizzled6）对牙髓干细胞（DPSCs）内皮分化的影响。通过增强型绿色荧光蛋白（EGFP）标记的慢病毒载体（短发夹 RNA-LRP6、短发夹 RNA-Frizzled6 或空载体对照）稳定转染 DPSCs。我们评估了 LRP6 和 Frizzled6 对受重组人 Wnt1（rhWnt1）和/或重组人血管内皮生长因子（rhVEGF）诱导的 DPSCs 表达内皮标记物和毛细血管管形成的影响。在体内，将沉默 LRP6、沉默 Frizzled6 或载体对照 DPSC 细胞接种到牙切片/支架中，并移植到免疫缺陷小鼠中。通过免疫组织化学法分析 EGFP 标记的 DPSCs 分化为血管内皮细胞后生成的血管密度。rhWnt1 和 rhVEGF 诱导对照 DPSCs 和 Frizzled6 沉默 DPSCs 中活性 β-连环蛋白的表达，但不诱导 LRP6 沉默 DPSCs 中活性 β-连环蛋白的表达。此外，LRP6 沉默 DPSCs 中 VEGF 和白细胞介素-8 的表达下调，但在对照 DPSCs 或 Frizzled6 沉默 DPSCs 中未下调（P <.05）。同样，rhWnt1 和 rhVEGF 诱导对照 DPSCs 和 Frizzled6 沉默 DPSCs 中血管内皮标记物 VEGF 受体-2 的表达，但不诱导 LRP6 沉默 DPSCs 中 VEGF 受体-2 的表达。这些数据与在 Matrigel 中，与对照 DPSCs 相比，沉默 LRP6 的 DPSCs 生成的毛细血管芽密度较低的趋势相关。在体内，与载体对照细胞相比，牙切片/支架中接种 DPSC-short hairpinRNA-LRP6 细胞的人血管密度（即 EGFP 阳性血管）较低（P <.05）。总的来说，这些数据表明 LRP6 信号对于人 DPSCs 的血管生成分化是必要的。
