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Angiopoietin-like 2 upregulation promotes human chondrocyte injury via NF-κB and p38/MAPK signaling pathway

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Abstract

Several cellular and molecular processes participate in the pathologic changes of osteoarthritis (OA). However, the core molecular regulators of these processes are unclear, and no effective treatment for OA disease has been developed so far. ANGPTL2 is well known for its tissue remolding and pro-inflammation properties. However, the role of ANGPTL2 in osteoarthritis (OA) still remains unclear. To explore the expression level of ANGPTL2 in human OA cartilage and investigate the function of ANGPTL2 in human chondrocytes injury, qRT-PCR, western blot and immunohistochemistry were employed to investigate the expression of ANGPTL2 between human OA and normal cartilage samples. Next, human primary chondrocytes were treated with IL-1β to mimic OA progress in vitro, and the expression of ANGPTL2 were tested by qRT-PCR and western blot. Furthermore, the effect of ANGPTL2 in the expression of pro-inflammation cytokines (IL-1β, IL-6), proteolytic enzymes (MMP-1, MMP-13) and component of the cartilage matrix (COL2A1 and aggrecan) in human primary chondrocyte were explored by gain-of-function and loss-of-function methods. Finally, the nuclear factor kappa B (NF-κB) and p38/MAPK signaling pathways were also tested by western blot analysis. In this study, firstly, the expression level of ANGPTL2 was elevated both in human OA cartilage samples and IL-1β stimulated human chondrocytes. Secondly, ANGPTL2 upregulation promotes extracellular matrix (ECM) degradation and inflammation mediator production in human chondrocytes. Finally, ANGPTL2 activated the NF-κB and p38/MAPK signaling pathways via integrin α5β1. This study, for the first time, highlights that ANGPTL2 secreted by human chondrocytes plays a negative role in the pathogenesis of osteoarthritis, and it may be a potential therapeutic target in OA.

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Abbreviations

ANGPTL2:

Angiopoietin-like 2

NF-κB:

Nuclear factor kB

MAPK:

Mitogen-activated protein kinase

OA:

Osteoarthritis

MMP:

Matrix metalloproteinase

ECM:

Extracellular matrix

IL:

Interleukin

ITGA5:

Integrin α5

ITGB1:

Integrin β1

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Acknowledgements

We thank Kai Lin, Yinglei Fang, Penghui Ke for assistance in collecting cartilage samples.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81672161) and National Undergraduate Traning Programs for Innovation and Entrepreneurship-China (No. 201810366025).

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Author notes

  1. Wenshan Shan, Chao Cheng and Wei Huang contributed equally to this work.

Authors and Affiliations

  1. Department of Orthopaedics, The First Affiliated Hospital of Anhui Medical University, 218#Ji Xi Road, Hefei, 230032, Anhui, China

    Wenshan Shan, Wei Huang, Zhenfei Ding, Guanjun Cui, Wei Lu, Fuen Liu & Zongsheng Yin

  2. School of Basic Medical Science, and the First Clinical Medical College, Anhui Medical University, 81# Mei Shan Road, Hefei, 230032, Anhui, China

    Sha Luo, JieGou Xu & Wei He

  3. Department of Orthopaedics, The Fourth Affiliated Hospital of Anhui Medical University, 372#Tun Xi Road, Hefei, 230032, Anhui, China

    Chao Cheng

  4. Division of Life Sciences and Medicine, Department of Orthopaedics, The First Affiliated Hospital of USTC, University of Science and Technology of China, 17#Lu Jiang Road, Hefei, 230001, Anhui, China

    Wei Huang

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  1. Wenshan Shan
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Contributions

Zongsheng Yin, Wei He and Jiegou Xu designed the study. Wenshan Shan, Zhenfei Ding, Guanjun Cui performed in vitro experiments. Chao Chen, Wei Huang, Wei Lu and Fuen Liu detected the expression level of ANGPTL2 in OA cartilage samples. Sha Luo participated in the supplementary experiment. Wenshan Shan wrote the paper.

Corresponding authors

Correspondence to JieGou Xu, Wei He or Zongsheng Yin.

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The authors declare that they have no competing interests.

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All cartilage samples were obtained from the First Affiliated Hospital of Anhui Medical University. This research was approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University.

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All patients obtained a written informed consent about the study.

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774_2019_1016_MOESM1_ESM.tif

Supplementary Fig. 1. Identification of human primary chondrocytes. SF1. A-C shows that type II collagen was high produced by human primary chondrocytes. Immunofluorescence of human primary chondrocytes with an anti-type II collagen antibody (SF1A). The nuclei of cells were revealed by the DAPI (SF1.B). Staining of human primary chondrocytes with toluidine blue, which reveals high production of proteoglycans in human chondrocytes (SF1.D). Scale bars (ABC) = 200 μm. Scale bars (D) = 100 μm (TIFF 77082 kb)

774_2019_1016_MOESM2_ESM.tif

Supplementary Fig. 2. Silencing of ANGPTL2 in chondrocyte. AB: Silencing of ANGPTL2 in vitro OA model Human chondrocytes were divided into four groups: Control, IL-1β (10 ng/mL), IL-1β (10 ng/ml) + siNC, IL-1β (10 ng/mL) + siANGPTL2. Chondrocytes were pretreated with siNC or siANGPTL2 for 24 h followed by stimulated with IL-1β (10 ng/ml) for 24 h, and then, RNA or protein was extracted from chondrocytes. (A) qRT-PCR and (B) Western Blot were used to evaluate the expression of ANGPTL2. CD: Silencing of ANGPTL2 in human primary chondrocyte without IL-1β. Human chondrocytes were divided into three groups: Control, siNC, siANGPTL2. Chondrocytes were pretreated with siNC or siANGPTL2 for 24 h and then, RNA or protein was extracted from chondrocytes. (A) Western Blot and (B) qRT-PCR were used to evaluate the expression of ANGPTL2. Results are expressed as mean ± SD. Welch Test followed by Games-Howell Test (Equal variances not assumed) (TIFF 9847 kb)

774_2019_1016_MOESM3_ESM.tif

Supplementary Fig. 3. Effects of ANGPTL2 on COL2A1 and aggrecan production in human primary chondrocytes. A-C Human chondrocytes were divided into four groups: Control, IL-1β (10 ng/mL), IL-1β (10 ng/ml) + siNC, IL-1β (10 ng/mL) + siANGPTL2. Chondrocytes were pretreated with siNC or siANGPTL2 for 24 h followed by stimulated with IL-1β (10 ng/ml) for 24 h, and then, RNA or protein was extracted from chondrocytes. (A)(B) qRT-PCR analysis for the mRNA expression levels of COL2A1 and aggrecan. (C)Western blots analysis for the protein expression of COL2A1 and aggrecan. D-F Human primary chondrocytes were treated with a serial dosage (0, 0.1, 0.2, 0.4 μg/ml) of rhANGPTL2 for 24 h. (D)(E) qRT-PCR analysis for the mRNA expression levels of COL2A1 and aggrecan. (F)Western blots analysis for the protein expression of COL2A1 and aggrecan. Results are expressed as mean ± SD. (D)Analysis of variance (ANOVA) followed by Bonferroni Test (equal variances). (A)Welch Test followed by Games-Howell Test (Equal variances not assumed). (BE) ANOVA Test (no significant) (TIFF 9789 kb)

774_2019_1016_MOESM4_ESM.tif

Supplementary Fig. 4. Effects of ANGPTL2 on caspase3 production in human primary chondrocytes. Human chondrocytes were divided into four groups: Control, IL-1β (10 ng/mL), IL-1β (10 ng/ml) + siNC, IL-1β (10 ng/mL) + siANGPTL2. Chondrocytes were pretreated with siNC or siANGPTL2 for 24 h followed by stimulated with IL-1β (10 ng/ml) for 24 h, and then, RNA or protein was extracted from chondrocytes. (A) Western Blot and (B) qRT-PCR were used to evaluate the expression of caspase3. Results are expressed as mean ± SD. ANOVA Test (no significant) (TIFF 4184 kb)

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Shan, W., Cheng, C., Huang, W. et al. Angiopoietin-like 2 upregulation promotes human chondrocyte injury via NF-κB and p38/MAPK signaling pathway. J Bone Miner Metab 37, 976–986 (2019). https://doi.org/10.1007/s00774-019-01016-w

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