The Significance of SDF-1α-CXCR4 Axis in in vivo Angiogenic Ability of Human Periodontal Ligament Stem Cells

Abstract

Periodontal ligament stem cells (PDLSCs) are multipotent stem cells derived from periodontium and have mesenchymal stem cell (MSC)-like characteristics. Recently, the perivascular region was recognized as the developmental origin of MSCs, which suggests the in vivo angiogenic potential of PDLSCs. In this study, we investigated whether PDLSCs could be a potential source of perivascular cells, which could contribute to in vivo angiogenesis. PDLSCs exhibited typical MSC-like characteristics such as the expression pattern of surface markers (CD29, CD44, CD73, and CD105) and differentiation potentials (osteogenic and adipogenic differentiation). Moreover, PDLSCs expressed perivascular cell markers such as NG2, αsmooth muscle actin, platelet-derived growth factor receptor β, and CD146. We conducted an in vivo Matrigel plug assay to confirm the in vivo angiogenic potential of PDLSCs. We could not observe significant vessel-like structures with PDLSCs alone or human umbilical vein endothelial cells (HU-VECs) alone at day 7 after injection. However, when PDLSCs and HUVECs were co-injected, there were vessel-like structures containing red blood cells in the lumens, which suggested that anastomosis occurred between newly formed vessels and host circulatory system. To block the SDF-1α and CXCR4 axis between PDLSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into the Matrigel plug. After day 3 and day 7 after injection, there were no significant vessel-like structures. In conclusion, we demonstrated the peri-vascular characteristics of PDLSCs and their contribution to in vivo angiogenesis, which might imply potential application of PDLSCs into the neovascularization of tissue engineering and vascular diseases.

Keywords: SDF-1α-CXCR4 axis; angiogenesis; mesenchymal stem cells; periodontal ligament stem cells; perivascular cells.

Fig. 1

Primary isolation and characterization of PDLSCs. Primarily isolated PDLSCs were cultured and characterized. (A) PDLSCs showed typical MSC-like morphology at the third passage. (B) The growth of PDLSCs was linear during the culture period. (C) The expression of surface markers was determined by FACS. PDLSCs were positive for MSC markers. PDLSCs were cultured in osteogenic or adipogenic differentiation medium for 21 days. (D, E) Calcium deposits and lipid vacuoles were stained by Alizarin red and Oil red O, respectively.

Fig. 2

The expression of perivascular cell markers in PDLSCs. The expression of perivascular cell markers in PDLSCs was determined by qPCR and FACS analysis. (A) The mRNA expression levels of NG2, α-SMA, PDGFRβ, and CD146 derived from three different lines of PDLSCs were shown as average ± standard deviation. The arbitrary unit on the Y-axis represented 2^−ΔCT × 10⁴. (B) In the results of FACS analysis, PDLSCs were positive for NG2, PDGFRβ, and CD146 with different expression levels of NG2, PDGFRβ, and CD146. One of representative data was shown.

Fig. 3

In vivo angiogenic potential of PDLSCs. To investigate the in vivo angiogenic potential of PDLSCs, in vivo Matrigel plug assay was performed. PDLSCs and HUVECs were subcutaneously injected separately or together into immunodeficient mice. At day 7 after injection, vessel-like structures in Matrigel plug were analyzed by H&E and immunofluorescent staining. (A) In the results of PDLSCs alone or HUVECs alone, there were no obvious vessel-like structures. However, when PDLSCs and HUVECs were co-injected, vessel-like structures were formed and red blood cells were observed in the lumen. (B) Immunofluorescent staining for CD31 and α-SMA revealed that vessel-like structures were formed by α-SMA-positive PDLSCs and CD31-positive HU-VECs.

Fig. 4

The involvement of the SDF-1α and CXCR4 axis in in vivo angiogenesis by PDLSCs and HUVECs. The expression of SDF-1α and CXCR4 was determined by qPCR. (A) PDLSCs expressed SDF-1α, but CXCR4 expression was not detected. In contrast, HUVECs expressed CXCR4, but SDF-1α expression was not detected. To confirm the functional involvement of the SDF-1α and CXCR4 axis in in vivo angiogenesis, AMD3100, a CXCR4 antagonist, was mixed with the Matrigel plug. (B) At day 3 after injection, there were no obvious vessel-like structures in the AMD3100-treated group compared to the control group. (C) At day 7 after injection, there were no vessel-like structures in the AMD3100-treated group compared to the control group.

Fig. 5

The effects of blocking the SDF-1α and CXCR4 axis on in vivo angiogenesis by PDLSCs and HUVECs. To investigate the localization of PDLSCs and HUVECs, immunofluorescent staining was conducted. Based on the results of immunofluorescent staining for CD31 and α-SMA, we confirmed that vessel-like structures were not formed in the AMD3100-treated group compared to the control group at day 3 and day 7 after injection (A and B, respectively).