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Angiopoietin-like protein 2 promotes inflammatory conditions in the ligamentum flavum in the pathogenesis of lumbar spinal canal stenosis by activating interleukin-6 expression

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Abstract

Purpose

Chronic inflammation is thought to cause ligamentum flavum (LF) degeneration and hypertrophy in lumbar spinal canal stenosis (LSCS). Angiopoietin-like protein 2 (Angptl2) is highly expressed in hypertrophied LF. Because Angptl2 regulates interleukin-6 (IL-6) expression in various tissues, we investigated whether IL-6 is expressed in hypertrophied LF and, if so, does Angptl2 induce IL-6 expression in LF fibroblasts.

Methods

LF tissue was obtained from LSCS patients and non-LSCS patients. Polymerase chain reaction (PCR) for Angptl2 and IL-6 genes and immunohistochemistry for IL-6 protein were performed in LF tissue. Fibroblasts from LF tissue were used for in vitro experiments. Expression of integrin α5β1 (an Angptl2 receptor) and Angptl2 binding to receptors on LF fibroblasts were examined by fluorescence-activated cell sorter analysis and cell adhesion assays. After Angptl2 recombinant protein treatment, NF-κB activation and IL-6 expression in LF fibroblasts were investigated by immunocytochemistry, PCR, and enzyme-linked immunosorbent assay.

Results

IL-6 mRNA expression was increased in hypertrophied LF tissue from LSCS patients and positively correlated with LF thickness and Angptl2 mRNA expression. IL-6 protein was highly expressed in LF fibroblasts in hypertrophied LF tissue. In vitro experiments demonstrated integrin α5β1 expression on LF fibroblasts and Angptl2 binding to cells via receptors. Angptl2 stimulation promoted NF-κB nuclear translocation and induced IL-6 expression and secretion in LF fibroblasts.

Conclusions

Angptl2 promotes inflammation in LF tissue by activating IL-6 expression, leading to LF degeneration and hypertrophy.

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Acknowledgments

We thank Ms. M. Nakata for technical assistance and Edanz for providing editorial assistance.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Department of Orthopaedic Surgery, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, 860-8556, Japan

    Takayuki Nakamura, Tatsuya Okada & Hiroshi Mizuta

  2. Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, 860-8556, Japan

    Takayuki Nakamura, Motoyoshi Endo & Yuichi Oike

  3. Nakamura Orthopaedic Clinic, 2-5-22 Ikeda-machi, Kita-ku, Kumamoto, 860-0082, Japan

    Takafumi Nakamura

  4. CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan

    Yuichi Oike

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  1. Takayuki Nakamura
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  2. Tatsuya Okada
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  4. Takafumi Nakamura
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Corresponding author

Correspondence to Takayuki Nakamura.

Electronic supplementary material

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586_2015_3835_MOESM1_ESM.tif

Supplementary material 1 (TIFF 1461 kb) S-Fig. 1 Angptl2 receptor expression in each LF fibroblast. Fluorescence-activated cell sorter analysis of LF fibroblasts for expression of integrin α5β1 (left) or leukocyte immunoglobulin-like receptor B2 (LILRB2) (right). Representative expression patterns obtained with anti-α5β1 and anti-LILRB2 antibodies (unfilled peaks) or isotype-matched immunoglobulin G (IgG) (shaded peaks) are shown. Analysis was conducted using Flow Jo software (Treestar, Inc., San Carlos, CA, USA)

586_2015_3835_MOESM2_ESM.tif

Supplementary material 2 (TIFF 1034 kb) S-Fig. 2 Quantification of the adhesion of LF fibroblasts to Angptl2. Quantification of the adhesion of LF fibroblasts (n 3) mediated via integrin α5β1. OD595 value of Angptl2-coating was set at 1. Data are expressed as the mean ± SEM. **p < 0.01 versus Angptl2 coating

586_2015_3835_MOESM3_ESM.tif

Supplementary material 3 (TIFF 6507 kb) S-Fig. 3 Immunocytochemistry for NF-κB p65 after Angptl2 administration in each cell. Note the nuclear translocation of p65 by Angptl2 and inhibition of the translocation by anti-integrin α5β1 antibody in LF fibroblasts. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI). Scale bars represent 50 μm in each panel

586_2015_3835_MOESM4_ESM.tif

Supplementary material 4 (TIFF 806 kb) S-Fig. 4 IL-6 expression by each LF fibroblasts in response to Angptl2. IL-6 mRNA expression in response to Angptl2 with or without BAY11-7085 (n = 3). The value of the controls (no Angptl2 treatment) was set at 1. Data are expressed as the mean ± SEM. **p < 0.01 compared with controls. †p < 0.01 compared with Angptl2 treatment

586_2015_3835_MOESM5_ESM.tif

Supplementary material 5 (TIFF 755 kb) S-Fig. 5 IL-6 secretion by each LF fibroblasts in response to Angptl2. IL-6 secretion (B) in response to Angptl2 with or without BAY11-7085 (n = 3). The value of the controls (no Angptl2 treatment) was set at 1. Data are expressed as the mean ± SEM. **p < 0.01 compared with controls. †p < 0.01 compared with Angptl2 treatment

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Nakamura, T., Okada, T., Endo, M. et al. Angiopoietin-like protein 2 promotes inflammatory conditions in the ligamentum flavum in the pathogenesis of lumbar spinal canal stenosis by activating interleukin-6 expression. Eur Spine J 24, 2001–2009 (2015). https://doi.org/10.1007/s00586-015-3835-z

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