1. Chondromodulin-1 ameliorates osteoarthritis progression by inhibiting HIF-2α activity 
X. Zhang, I. Prasadam, W. Fang, R. Crawford, Y. Xiao 
Osteoarthritis and Cartilage 
Volume 24, Issue 11, Pages (November 2016)
DOI: /j.joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Imbalance of angiogenic and anti-angiogenic factors in OA cartilage. The figure is to show the imbalanced distribution of angiogenic and anti-angiogenic factors in OA cartilage. (A) Immunohistochemical staining of ChM-1 and VEGFA in human OA cartilage. Scale bars = 100 μm. Representative images of experiments performed on samples collected from five donors. (B, C) Protein and gene expression of anti-angiogenic factors (ChM-1, TIMP-1 and TIMP-2) and hypertrophic marker RUNX2 in human OA cartilage tissues. Experiments performed on three donors. (D) Human angiogenesis qRT-PCR array performed on cartilage samples collected and graded according to the disease severity. Columns represent fold changes of individual gene expression between severe OA cartilage and mild OA cartilage. Upper columns (>1.00) represent up-regulated genes, lower columns (<1.00) represent down-regulated genes. Graph is one representative data from two independent experiments performed on individual donors.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Expression of chondrogenic, angiogenic and anti-angiogenic markers during re-differentiation of ACCs and differentiation of BMSCs. (A) Immunohistochemical staining of ChM-1 and a time-dependent qRT-PCR analysis of ChM-1, VEGFA and COL2A1 gene expression in pellet culture of ACCs for 1, 2 and 3 weeks. Values are mean ± s.e.m (Results shown are performed on three donor samples with three technical replicates. *P < 0.05 vs week 1. (B) Safranin-O staining and immunohistochemical staining of COL2A1, ChM-1 and VEGFA in pellet culture of ACCs for 3-weeks. Gene expression of chondrogenic markers (COL2A1, ACAN and SOX9), anti-angiogenic and angiogenic-related factors (ChM-1, Endostatin and VEGFA) was measured by qRT-PCR. Scale bars = 100 μm. Values are mean ± s.e.m. Results shown are performed on three donors with three technical replicates each time. *P < 0.05 vs ACCs group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Effects of gain and loss of function of ChM-1 on hypertrophic markers in primary cultured ACCs from healthy cartilage. (A) mRNA and protein expression of RUNX2, COL10A1, MMP-13, and VEGFA in ACCs with ChM-1 overexpression. ACCs were transfected with LV-ChM-1 or mock lentivirus (LV-GFP) and exposed to TNF-α (10 ng/mL) for 48 h. (B) Analyses of the hypertrophic markers in ACCs transfected with ChM-1 siRNA (100 nM) or scrambled siRNA, followed by treatment with TNF-α (10 ng/mL) for 48 h. Non-transfected ACCs serves as empty control for both (A) and (B). Values are mean ± s.e.m. Results shown are performed on three donors with three technical replicates each time. *P < 0.05 vs LV-GFP group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Overexpression of ChM-1 in articular cartilage delayed OA progression in MSX rat model. (A) Gross observation and Safranin-O staining of tibia cartilage section in surgical MSX OA rat model with intra-articular injection of LV-ChM-1 or LV-GFP for 5 weeks. Transfection efficacy confirmed by GFP and V5-tag fluorescence and ChM-1 staining. Scale bars = 50 μm. (B) Immunohistochemical staining of chondrogenic marker (COL2A1 and aggrecan) and hypertrophic markers (MMP-13, COL10A1 and VEGFA) in rat OA cartilage injected with LV-ChM-1 or LV-GFP. Arrows means the immune-positive cells with indicated antibodies. Scale bars = 100 μm. (C) Quantification of OA progression by Mankin scoring system. (D) Gene expression of ChM-1 and hypertrophic markers in rat cartilage injected with LV-ChM-1 or LV-GFP. Values represent mean ± s.e.m, Representative images from experiments performed on six individual animals. *P < 0.05 vs LV-GFP group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
Expression of HIF-2α in vivo and in vitro. (A) Immunohistochemical staining of HIF-2α in rat normal and OA cartilage. Scale bars = 100 μm. (B) HIF-2α protein expression in chondrocyte cell line C28/I2 cells under the stimulation of TNF-α (1, 10 or 50 ng/mL) or hypoxia condition (1% O2) for indicated time-point. GAPDH was included as a loading control. (C) Western blot and qRT-PCR analysis of HIF-2α expression in C28/I2 cells transfected with LV-ChM-1 or LV-GFP and exposed to 10 ng/mL TNF-α for indicated time-point. Values represent mean ± s.e.m. Results shown are performed on three donors with three technical replicates. *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
ChM-1 inhibits chondrocyte hypertrophy through regulation of HIF-2α activity. (A) Cytoplasmic and nuclear abundance of HIF-2α in C28/I2 cells with ChM-1 overexpression demonstrated by Western blot. C28/I2 cells were transfected with LV-ChM-1 or LV-GFP and exposed to TNF-α for indicated time-point, and then cytoplasmic and nuclear fractions were extracted. Tubulin and PCNA serve as a loading control for cytoplasmic and nuclear protein, respectively. The intensities of the bands were normalized to loading control and then further converted to the fold change of the first time-point. (B) Intra-cellular localization of HIF-2α in C28/I2 cells with ChM-1 overexpression demonstrated by immunofluorescence staining. C28/I2 cells were transfected with LV-ChM-1 and exposed to 10 ng/mL TNF-α for 1 h. DAPI was used to visualize nuclei. Non-transfected ACCs serves as control. Scale bars, 50 μm. Arrow head, positive nuclear staining of HIF-2α; Arrow, negative nuclear staining of HIF-2α. (C) ChIP assay for detection the DNA binding activity of HIF-2α to the promoter of hypertrophic genes of COL10A1, MMP-13 and VEGFA in C28/I2 cells with ChM-1 overexpression. C28/I2 cells were transfected with LV-ChM-1 or LV-GFP and exposed to TNF-α for 6 h. Non-transfected ACCs serves as empty control. HRE = hypoxia-responsive element. TSS = transcription start site. Values are mean ± s.e.m. Results shown are performed on three donors with three replicates. *P < 0.05 vs LV-GFP group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

8. Fig. 7
Schematic illustration showing the protective effect of ChM-1 on cartilage homeostasis and the underlying mechanisms. In normal cartilage, the abundant expression of ChM-1 blocks the angiogenic and hypertrophic activity of HIF-2α; whereas in OA cartilage, lack of ChM-1 results in activating transcriptional activity of HIF-2α, resulting in chondrocyte hypertrophy and cartilage degradation through the enhancement expression of catabolic genes of COL10A1, MMP-13 and VEGFA. ChM-1 supplementation might be a potential therapeutic modality in the treatment of OA.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

9. Osteoarthritis and Cartilage 2016 24, 1970-1980DOI: (10. 1016/j. joca
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10. Osteoarthritis and Cartilage 2016 24, 1970-1980DOI: (10. 1016/j. joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

11. Osteoarthritis and Cartilage 2016 24, 1970-1980DOI: (10. 1016/j. joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions