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. 2017 Mar 30;8(3):e2715.
doi: 10.1038/cddis.2017.129.

VSports app下载 - TNF-α-induced LRG1 promotes angiogenesis and mesenchymal stem cell migration in the subchondral bone during osteoarthritis

Yiyun Wang  1 , Jiajia Xu  1 , Xudong Zhang  1 , Chuandong Wang  1 , Yan Huang  1 , Kerong Dai  1   2 , Xiaoling Zhang  1   2
Affiliations

TNF-α-induced LRG1 promotes angiogenesis and mesenchymal stem cell migration in the subchondral bone during osteoarthritis

Yiyun Wang et al. Cell Death Dis. .

Abstract

The incomplete understanding of aberrant neovascularization, which contributes to osteoarthritis suggests that additional modulators have yet to be identified. Our objective was to identify the role of Leucine-rich-alpha-2-glycoprotein1 (LRG1), a new regulator of pathogenic angiogenesis, in osteoarthritis progression and to develop effective treatment strategies. In this study, immunohistochemistry showed that LRG1 was increased in the subchondral bone and articular cartilage in anterior cruciate ligament transection (ACLT) mice. Further studies were focused on the role of LRG1 in osteoarthritis. Results showed that LRG1 promoted angiogenesis and mesenchymal stem cells (MSC) migration, which contribute to aberrant bone formation in the subchondral bone. Moreover, tumor necrosis factor-α (TNF-α), not interleukin-1β (IL-1β), IL-6 or IL-17, induced the LRG1 expression in human umbilical vein endothelial cells and this effect was inhibited by p38 mitogen-activated protein kinase or NF-κB inhibitor. Notably, inhibition of TNF-α and LRG1 activity by Lenalidomide, an inhibitor of TNF-α production, in ACLT mice attenuated degeneration of osteoarthritis articular cartilage. This study shows that TNF-α is the predominant proinflammatory cytokine that induces the secretion of LRG1. LRG1 contributes to angiogenesis-coupled de novo bone formation by increasing angiogenesis and recruiting MSCs in the subchondral bone of osteoarthritis joints. Inhibition of TNF-α and LRG1 by Lenalidomide could be a potential therapeutic approach VSports手机版. .

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
LRG1 was upregulated in the subchondral bone and articular cartilage and associated with angiogenesis in ACLT mice. (a and b) Immunohistochemical analysis results of LRG1 in mouse tibial subchondral bone (top) and articular cartilage (bottom) collected 30 days after ACLT surgery (a) and collected 60 days after ACLT surgery (b); n=5 per group. Scale bars, 20 μm. (c) Immunohistochemical analysis of CD31 in mouse tibial subchondral bone; n=5 per group. Scale bars, 20 μm. (d) Matrigel tube-formation assay images (left) and quantitative analysis of cumulative tube length (right). Scale bar, 50 μm. Values are given as means±S.D. (three independent experiments), *P<0.05
Figure 2
Figure 2
LRG1 had no direct effect on osteogenesis. (a and b) Immunohistochemical analysis of nestin (a) and osterix (b) in mouse tibial subchondral bone collected 30 days after ACLT surgery, n=5 per group. Scale bars, 20 μm. (c) The cell proliferation was determined in hBMMSCs by PrestoBlue. (d) Alizarin Red S staining (left) of cultured hBMMSCs after treatment with osteogenic medium plus LRG1. Quantitative of Alizarin Red S staining showed the levels of mineralization (right). CM, control medium; OM, osteogenesis medium; OM+LRG1, osteogenesis medium plus LRG1. (e) Relative expression levels of osteoblast markers in hBMMSCs indicated in d were quantified by RT-PCR. All values are given as means±S.D. (three independent experiments); *P<0.05, **P<0.01
Figure 3
Figure 3
LRG1 induced hBMMSCs migration. (a and b) Transwell assay images (a) and quantitative analysis (b) for the migration of hBMMSCs by using mediums plus different concentrations of LRG1. Scale bar, 100 μm. (c) Western blots of the p38 phosphorylation in hBMMSCs treated with LRG1 for 15 min. The relative expression levels of protein are shown at the bottom of the bands as normalized by the total p38 level. (d and e) Transwell assay images (d) and quantitative analysis (e) for the migration of hBMMSCs that were preincubated with vehicle or p38 inhibitor (inh p38) by using indicated mediums. Scale bar, 100 μm. All values are given as means±S.D. (three independent experiments); *P<0.05, **P<0.01
Figure 4
Figure 4
TNF-α induced LRG1 secretion in HUVECs through p38 and p65 signaling. (a) RT-PCR analysis of LRG1 in HUVECs, stimulated with IL-1β, TNF-α, IL-6 and IL-17 (10 or 50 ng/ml). (b) Western blot analysis of LRG1 in HUVECs stimulated with TNF-α in different concentrations. The relative expression levels of protein are shown at the bottom of the bands as normalized by the GAPDH level. (c) Knockdown of LRG1 expression in HUVECs by siRNA. (d) Matrigel tube-formation assay of HUVECs after treatment with supernatants, which were collected from siLRG1 or siNC transfected HUVEC cultures in the presence of TNF-α. Images (left) and quantitative analysis of cumulative tube length (right). Scale bar, 20 μm. (e) Transwell assays of hBMMSCs after treatment with supernatants, which were collected from siLRG1 or siNC transfected HUVEC cultures in the presence of TNF-α. Images (left) and quantitative analysis (right). Scale bar, 100 μm. (f) Western blot analysis of the phosphorylation of p38 and p65 in HUVECs treated with TNF-α at different times. The relative expression levels of protein are shown at the bottom of the bands as normalized by the total p38 and p65 levels, respectively. (g and h) RT-PCR (g) or western blot analysis of LRG1 and phosphorylation of p38 and p65 (h) in HUVECs stimulated with TNF-α and pretreated or not with p38 inhibitor (p38 inh) and p65 inhibitor (p65 inh). The relative expression levels of protein are shown at the bottom of the bands. LRG1 level is normalized by the GAPDH level, and the phosphorylation of p38 and p65 levels are normalized by the total p38 and p65 levels, respectively. All values are given as means±S.D. (three independent experiments); *P<0.05, **P<0.01
Figure 5
Figure 5
LEN, a TNF-α production inhibitor, reduced LRG1 expression, alleviated the changes in the subchondral bone, and attenuated articular cartilage degeneration in ACLT. (a and b) Immunohistochemical analysis of LRG1 (top, a), CD31 (middle, a), TNF-α (bottom, a), nestin (Top, b) and osterix (bottom, b) in tibial subchondral bone harvested 30 days after sham operation (sham), ACLT operation and treatment with vehicle (vehicle) or ACLT operation and treatment with LEN (LEN); n=5 per group. Scale bars, 20 μm. (c) Safranin O and fast green staining in the articular cartilage of mice 60 days after surgery; n=5 per group. Scale bars, 50 μm. (d) OARSI scores in c. (e) Immunohistochemical analysis of MMP13 (top) and LRG1 (bottom) in the articular cartilage of mice 30 days after surgery; n=5 per group. Scale bars, 20 μm. All values are given as means±S.D. **P<0.01 compared with the sham-operated group; #P<0.05 compared with the vehicle-treated ACLT group
Figure 6
Figure 6
Proposed role of LRG1 in OA. The elevated TNF-α induces LRG1 secretion. LRG1 promotes angiogenesis coupling with de novo bone formation by recruiting MSCs in the subchondral bone of OA joints
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