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. 2018 Dec 17;13(12):e0209300.
doi: 10.1371/journal.pone.0209300. eCollection 2018.

Angiopoietin-2 promotes osteogenic differentiation of thoracic ligamentum flavum cells via modulating the Notch signaling pathway

Xiaoxi Yang  1 , Zhongqiang Chen  1 , Xiangyu Meng  2 , Chuiguo Sun  1 , Mengtao Li  2 , Li Shu  2 , Dongwei Fan  1 , Tianqi Fan  1 , Ann Y Huang  3 , Chi Zhang  2   4   5
Affiliations

Angiopoietin-2 promotes osteogenic differentiation of thoracic ligamentum flavum cells via modulating the Notch signaling pathway

Xiaoxi Yang et al. PLoS One. .

Abstract

Thoracic ossification of the ligamentum flavum (TOLF) is heterotopic ossification of spinal ligaments, which may cause serious thoracic spinal canal stenosis and myelopathy. However, the underlying etiology remains inadequately understood. In this study, the ossification patterns of TOLF were analyzed by micro-computer tomography (micro-CT). The expression profile of genes associated with angiogenesis was analyzed in thoracic ligamentum flavum cells at sites of different patterns of ossification using RNA sequencing. Significant differences in the expression profile of several genes were identified from Gene Ontology (GO) analysis. Angiopoietin-2 (ANGPT2) was significantly up-regulated in primary thoracic ligamentum flavum cells during osteogenic differentiation. To address the effect of ANGPT2 on Notch signaling and osteogenesis, ANGPT2 stimulation increased the expression of Notch2 and osteogenic markers of primary thoracic ligamentum flavum cells of immature ossification, while inhibition of ANGPT2 exhibited opposite effect on Notch pathway and osteogenesis of cells of mature ossification. These findings provide the first evidence for positive regulation of ANGPT2 on osteogenic differentiation in human thoracic ligamentum flavum cells via modulating the Notch signaling pathway.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Comparison between immature ossification (imOS) and mature ossification (mOS) of TOLF.
(A) Micro-CT images of immature ossification (imOS) and mature ossification (mOS) of TOLF; (B) morphological parameters; * P< 0.05 compared with imOS; (C) Volcano plots showing the DEGs among imOS and OS; (D) Differential GO terms distribution.
Fig 2
Fig 2. Gene activity comparison between immature ossification (imOS) and mature ossification (mOS) of TOLF.
(A) Expression level of genes in primary thoracic ligamentum flavum cells. Expressions of ANGPT2, Notch1 and Notch2 were assessed via qRT-PCR.* P< 0.05 compared with imOS; (B) Protein levels in primary thoracic ligamentum flavum cells. Proteins of ANGPT2, Notch1 and Notch2 were analyzed via Western blot; (C) Osteogenic markergene expressionsin primary thoracic ligamentum flavum cells via qRT-PCR. * P< 0.05 compared with imOS; (D) Osteogenic marker protein levels in primary thoracic ligamentum flavum cells via Western blot; (E) ALP staining of ligamentum flavum cells from imOS and mOS patterns; scale bar represents 200 μm.
Fig 3
Fig 3. ANGTP2 stimulation enhances notch signaling and osteogenic differentiation of ligamentum flavum cells.
(A) Effect of ANGPT2 on Notch1 and Notch2 expressions. Notch1 and Notch2 expressions were assessed via qRT-PCR in imOS ligamentum flavum cells with ANGPT2 stimulation (St.); * P< 0.05 compared with no stimulation group (Non-St.); (B) Effect of ANGPT2 on protein levels of Notch1 and Notch2 via Western blot; (C) Osteogenic marker expressions after ANGPT2 stimulation via qRT-PCR. * P< 0.05 compared with Non-St; (D) Effect of ANGPT2 on protein levels of osteogenic markers; (E) ALP staining following ANGPT2 stimulation; scale bar represents 200 μm.
Fig 4
Fig 4. Effects of ANGTP2 knockdown on notch signaling and osteogenic differentiation of ligamentum flavum cells.
(A) Effect of siANGPT2 on Notch1 and Notch2 expressions. ANGPT2, Notch1 and Notch2 expressions were assessed via qRT-PCR in mOS ligamentum flavum cells afterANGPT2 siRNA transfection (siANGPT2); * P< 0.05 compared with NC. (B) Effect of siANGPT2 on protein levels of Notch1 and Notch2 via Western blot; (C) Osteogenic marker expressions after siANGPT2 transfection via qRT-PCR.; * P< 0.05 compared with NC; (D) Effect of siANGPT2 on protein levels of osteogenic markers; (E) ALP staining following siANGPT2 transfection; scale bar represents 200μm; (F) Effect of Notch2 siRNA transfection (siNotch2) on ANGPT2 expression; * P< 0.05 compared with NC; (G) Effect of siNotch2 on ANGPT2 protein level.
See this image and copyright information in PMC

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