Lipoprotein Receptor-related Protein 6 Signaling is Necessary for Vasculogenic Differentiation of Human Dental Pulp Stem Cells

Abstract

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 factor₁₆₅ (rhVEGF₁₆₅). 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 rhVEGF₁₆₅ 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 rhVEGF₁₆₅ 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.

Keywords: Angiogenesis; Wnt; cell fate; regenerative endodontics; tissue engineering.

Figure 1

Effect of LRP6 and Frizzled6 on VEGF or Wnt1 signaling in DPSC cells. (A) Western blots to evaluate the effectiveness of LRP6 or Frizzled6 silencing in DPSC cells. We tested several shRNA sequences for Frizzled6 (#30,31,33,52) and for LRP6 (#1–5). (B) Fluorescence image to evaluate the effectiveness of lentivirus-mediated transduction of shRNA-LRP6 or shRNA-Frizzled6 into DPSC cells. All lentiviral vectors used here (including controls) contained EGFP. Photomicrographs were taken at 200× magnification. (C) Western blots depicting effect of increasing concentrations of rhVEGF₁₆₅ or rhWnt1 on phosphorylated and total β-catenin, GSK-3β, and AKT.

Figure 2

LRP6 silencing inhibits vasculogenic differentiation of DPSC cells in vitro. (A) Western blots depicting protein expression of endothelial markers (VEGFR1, VEGFR2) upon culture of DPSC in control medium (alpha-MEM), endothelial growth medium (EGM2-MV), or EGM2-MV supplemented with 50 ng/ml rhVEGF₁₆₅ or rhWnt1. (B,C) Graphs depicting the results of ELISA for VEGF (B) and IL-8 (C) in DPSC cells cultured in control alpha-MEM medium. Different letters represent p<0.05.

Figure 3

Effect of LRP6 or Frizzled6 signaling on capillary sprouting of DPSC in 3-D cultures. (A) Photomicrographs of capillary tube networks generated by human dermal microvascular endothelial cells (HDMEC) or by DPSC seeded in Matrigel and exposed to EGM2-MV supplemented with 50 ng/ml rhVEGF₁₆₅ for up to 144 hours (200×). (B) Graph depicting the number of sprouts/microscopic field generated by HDMEC, DPSC-silenced cells, or controls seeded on Matrigel and stimulated with EGM2-MV supplemented with 50 ng/ml rhVEGF₁₆₅ for up to 144 hours.

Figure 4

LRP6 silencing inhibits endothelial differentiation of DPSC in vivo. (A) Tooth slices/scaffolds seeded with LRP6-silenced DPSC, Frizzled6-silenced DPSC, or vector control DPSC were transplanted into the subcutaneous space of immunodeficient mice. After 28 days, tooth slice/scaffolds were retrieved, fixed, and analyzed by hematoxilin-eosin staining (200×), or immunohistochemistry with EGFP antibody (400×). (B) Graph depicting the density of EGFP-positive blood vessels (i.e. generated by DPSC cells) in 6 tooth slice/scaffolds per experimental condition. Blood vessels were counted in 10 microscopic fields/specimen at 200× magnification. Different letters represent p<0.05.