Yaoyao Du, Yue Wang, Li Wang, Bo Liu, Qingyun Tian, Chuan-ju Liu, Tao Zhang, Qingbo Xu, Yi Zhu, Oldberg Ake, Yongfen Qi, Chaochu Tang, Wei Kong and Xian Wang
Circulation Research. 2011;108:917-928, originally published April 14, 2011
Yaoyao Du, Yue Wang, Li Wang, Bo Liu, Qingyun Tian, Chuan-ju Liu, Tao Zhang, Qingbo Xu, Yi Zhu, Oldberg Ake, Yongfen Qi, Chaochu Tang, Wei Kong, Xian Wang
Circulation Research April 2011, 108 (8) 917-928; DOI: https://doi.org/10.1161/CIRCRESAHA.110.234328
By YaoyaoDu, YueWang, LiWang, BoLiu, QingyunTian, Chuan-juLiu, TaoZhang, QingboXu, YiZhu, OldbergAke, YongfenQi, ChaochuTang, WeiKong and XianWang
COMP protein level is decreased in calcifying VSMCs and arteries. A, Representative Western blot and quantitative analysis of COMP protein in bovine V...Show More
COMP protein level is decreased in calcifying VSMCs and arteries. A, Representative Western blot and quantitative analysis of COMP protein in bovine VSMCs cultured in calcifying medium containing 10 mmol/L β-GP for 3, 5, and 7 days. Relative COMP protein level was normalized to that of β-actin and expressed as means±SEM from 3 independent experiments performed in duplicate (bottom). *P<0.05. B, COMP protein level in A7r5 cells exposed to 5 mmol/L CaCl2 for 3, 6, 9, and 12 days. C, Representative Western blot and quantitative analysis of COMP protein in abdominal aortas from sham-operated or rats with CRF (5/6 nephrectomization plus high phosphate diet) at time of euthanasia (n=7 per group). *P<0.05 vs sham control. D, COMP expression in rat abdominal aortas periadventitially treated with 0.2 mol/L NaCl or CaCl2 (n=5 per group). *P<0.05.Show Less
Yaoyao Du, Yue Wang, Li Wang, Bo Liu, Qingyun Tian, Chuan-ju Liu, Tao Zhang, Qingbo Xu, Yi Zhu, Oldberg Ake, Yongfen Qi, Chaochu Tang, Wei Kong, Xian Wang
Circulation Research April 2011, 108 (8) 917-928; DOI: https://doi.org/10.1161/CIRCRESAHA.110.234328
By YaoyaoDu, YueWang, LiWang, BoLiu, QingyunTian, Chuan-juLiu, TaoZhang, QingboXu, YiZhu, OldbergAke, YongfenQi, ChaochuTang, WeiKong and XianWang
COMP deficiency aggravates VSMCs calcification in vitro. A, Quantitative analysis of calcium deposition. A7r5 cells were transfected with scramble siR...Show More
COMP deficiency aggravates VSMCs calcification in vitro. A, Quantitative analysis of calcium deposition. A7r5 cells were transfected with scramble siRNA or COMP siRNA 48 hours before incubation with calcifying medium containing 5 mmol/L CaCl2 for an additional 12 days. B, A7r5 cells were stained for mineralization by Alizarin red S. Scale bar, 20 μm. C, Identification of COMP deficiency in VSMCs of COMP knockout mice or littermate wild-type c57 mice by Western blot analysis. D, Quantitative analysis of calcium deposition in VSMCs from COMP knockout or wild-type mice in response to high phosphate for 12 days. Values are expressed as means±SEM from 3 independent experiments. *P<0.05. E, Representative Alizarin red S staining of calcium nodule. Scale bar, 20 μm.Show Less
Yaoyao Du, Yue Wang, Li Wang, Bo Liu, Qingyun Tian, Chuan-ju Liu, Tao Zhang, Qingbo Xu, Yi Zhu, Oldberg Ake, Yongfen Qi, Chaochu Tang, Wei Kong, Xian Wang
Circulation Research April 2011, 108 (8) 917-928; DOI: https://doi.org/10.1161/CIRCRESAHA.110.234328
By YaoyaoDu, YueWang, LiWang, BoLiu, QingyunTian, Chuan-juLiu, TaoZhang, QingboXu, YiZhu, OldbergAke, YongfenQi, ChaochuTang, WeiKong and XianWang
COMP overexpression inhibits VSMC calcification in vitro. Quantification of calcium deposition and Alizarin red S staining in bovine VSMCs (A and B) o...Show More
COMP overexpression inhibits VSMC calcification in vitro. Quantification of calcium deposition and Alizarin red S staining in bovine VSMCs (A and B) or rat A7r5 cells (C and D). Bovine VSMCs were infected with 5 multiplicities of infection of Ad-GFP or Ad-COMP for 48 hours before β-GP stimulation for an additional 7 days. Similarly, A7r5 cells were infected with 50 multiplicities of infection of Ad-GFP or Ad-COMP, followed by administration of 5 mmol/L CaCl2 for 12 days. The results are means±SEM from 3 independent experiments performed in duplicate. *P<0.05. The reddish/purple staining indicates mineral deposition. Scale bar, 20 μm.Show Less
Yaoyao Du, Yue Wang, Li Wang, Bo Liu, Qingyun Tian, Chuan-ju Liu, Tao Zhang, Qingbo Xu, Yi Zhu, Oldberg Ake, Yongfen Qi, Chaochu Tang, Wei Kong, Xian Wang
Circulation Research April 2011, 108 (8) 917-928; DOI: https://doi.org/10.1161/CIRCRESAHA.110.234328
By YaoyaoDu, YueWang, LiWang, BoLiu, QingyunTian, Chuan-juLiu, TaoZhang, QingboXu, YiZhu, OldbergAke, YongfenQi, ChaochuTang, WeiKong and XianWang
Overexpression of COMP retards aortic calcification. A, Rat abdominal aortas were infected periadventitially with Ad-GFP or Ad-COMP. After 3 days, abd...Show More
Overexpression of COMP retards aortic calcification. A, Rat abdominal aortas were infected periadventitially with Ad-GFP or Ad-COMP. After 3 days, abdominal aortas were cut into rings and cultured in vitro in DMEM or calcifying medium containing 3.8 mmol/L PO43− for 6 days. A, Quantification of calcium deposition. B, von Kossa and hematoxylin/eosin staining of mineral nodules. Values are means±SEM (n=6 for each group). *P<0.05. C, Rat abdominal arteries were treated with 0.2 mol/L CaCl2 for 10 minutes before periadventitial infection with Ad-GFP or Ad-COMP. After 7 days, rats were euthanized and abdominal arteries were isolated and analyzed for calcium deposition (C) and von Kossa staining (D). *P<0.05. Scale bar, 10 μm.Show Less
Yaoyao Du, Yue Wang, Li Wang, Bo Liu, Qingyun Tian, Chuan-ju Liu, Tao Zhang, Qingbo Xu, Yi Zhu, Oldberg Ake, Yongfen Qi, Chaochu Tang, Wei Kong, Xian Wang
Circulation Research April 2011, 108 (8) 917-928; DOI: https://doi.org/10.1161/CIRCRESAHA.110.234328
By YaoyaoDu, YueWang, LiWang, BoLiu, QingyunTian, Chuan-juLiu, TaoZhang, QingboXu, YiZhu, OldbergAke, YongfenQi, ChaochuTang, WeiKong and XianWang
COMP negatively regulates osteochondrogenic transition of VSMCs. Quantification of relative mRNA level of osteogenic-related marker genes (A) and smoo...Show More
COMP negatively regulates osteochondrogenic transition of VSMCs. Quantification of relative mRNA level of osteogenic-related marker genes (A) and smooth muscle marker genes (B) in the absence or presence of COMP silencing. *P<0.05 vs scramble siRNA. C, Quantitative real-time PCR analysis of osteogenic markers in VSMCs incubated with calcifying medium containing CaCl2 for 12 days. VSMCs were infected with Ad-GFP or Ad-COMP, respectively, before CaCl2 administration. *P<0.05. D, Representative Western blot and quantification analysis of expression of osteogenic markers (Runx2 and BMP-2) and smooth muscle lineage markers (α-actin and SM 22α) in rat abdominal aortic rings treated with 3.8 mmol/L Pi for 3 days after infection with Ad-GFP or Ad-COMP (n=5 per group). *P<0.05. E, Representative Western blot and quantification of osteogenic and SMC lineage protein expression in CaCl2-treated rat abdominal arteries infected with Ad-GFP or Ad-COMP at 7 days (n=5 per group). *P<0.05.Show Less
Yaoyao Du, Yue Wang, Li Wang, Bo Liu, Qingyun Tian, Chuan-ju Liu, Tao Zhang, Qingbo Xu, Yi Zhu, Oldberg Ake, Yongfen Qi, Chaochu Tang, Wei Kong, Xian Wang
Circulation Research April 2011, 108 (8) 917-928; DOI: https://doi.org/10.1161/CIRCRESAHA.110.234328
By YaoyaoDu, YueWang, LiWang, BoLiu, QingyunTian, Chuan-juLiu, TaoZhang, QingboXu, YiZhu, OldbergAke, YongfenQi, ChaochuTang, WeiKong and XianWang
COMP inhibits BMP-2 osteogenic signaling. A, Quantitative real-time PCR analysis of osteogenic gene expression in VSMCs transfected with scramble or C...Show More
COMP inhibits BMP-2 osteogenic signaling. A, Quantitative real-time PCR analysis of osteogenic gene expression in VSMCs transfected with scramble or COMP siRNA in the presence or absence of soluble BMPR-IA (1 mg/L). B, Relative expression of endogenous BMP-2 mRNA in VSMCs in response to treatment with increasing amounts of exogenous BMP-2 for 24 hours. *P<0.05 compared with control. C, Western blot analysis of BMP-2–induced Smad1/5/8 activation and downstream Runx2/Msx2 expression in VSMC. A7r5 cells were transfected with scramble or COMP-specific siRNA. Forty-eight hours later, Smad1/5/8 activation was detected in VSMCs stimulated by recombinant human BMP-2 (500 μg/L) for an additional 30 minutes. Expression level of Runx2 and Msx2 were assessed in VSMCs stimulated by BMP-2 for 24 hours. D, Western blot and quantitative analysis of BMP-2–induced Smad1/5/8 activation. A7r5 cells were infected with Ad-GFP or Ad-COMP 48 hours before challenge with recombinant human BMP-2 (500 μg/L) for 30 minutes. E and F, Relative mRNA (E) and protein (F) level of BMP-2–induced osteogenic transcriptional factors Runx2, Msx2, and osteocalcin (OCN). A7r5 cells were infected with Ad-GFP or Ad-COMP for 48 hours before treatment with recombinant BMP-2 (500 μg/L) for 3 hours (for mRNA) or 24 hours (for protein), respectively.Show Less
Yaoyao Du, Yue Wang, Li Wang, Bo Liu, Qingyun Tian, Chuan-ju Liu, Tao Zhang, Qingbo Xu, Yi Zhu, Oldberg Ake, Yongfen Qi, Chaochu Tang, Wei Kong, Xian Wang
Circulation Research April 2011, 108 (8) 917-928; DOI: https://doi.org/10.1161/CIRCRESAHA.110.234328
By YaoyaoDu, YueWang, LiWang, BoLiu, QingyunTian, Chuan-juLiu, TaoZhang, QingboXu, YiZhu, OldbergAke, YongfenQi, ChaochuTang, WeiKong and XianWang
COMP associates with BMP-2 and inhibits BMP-2 receptor binding. A, Co-IP assay in HEK293 cell line stably transfected with COMP. Recombinant human BMP...Show More
COMP associates with BMP-2 and inhibits BMP-2 receptor binding. A, Co-IP assay in HEK293 cell line stably transfected with COMP. Recombinant human BMP-2 (500 μg/L) was added and incubated for 12 hours. Left, Whole-cell lysates of HEK293 cells were immunoprecipitated with anti–BMP-2 antibody or control IgG before protein A agarose beads, then analyzed by Western blot (WB) with anti-COMP antibody. Right, HEK293 cell extracts were immunoprecipitated with anti-COMP antibodies or control IgG and analyzed by Western blot with anti–BMP-2 antibody. B, Co-IP assay of rat VSMCs and aortas. Left, VSMCs lysates or vascular extracts were incubated with anti–BMP-2 antibody or control IgG, then protein A agarose, and then analyzed by Western blot with anti-COMP antibody. Right, VSMCs lysates or vascular extracts were immunoprecipitated with anti-COMP antibodies or control IgG and analyzed by Western blot with anti–BMP-2 antibody. C, Solid phase binding assay. Various amounts of recombinant human BMP-2 or purified COMP underwent 10% SDS-PAGE and were incubated with purified COMP or human recombinant BMP-2. Anti-COMP or anti–BMP-2 antibodies were used for subsequent detection. BSA served as negative control. D, BMP-2 selectively binds to the C-terminal domain of COMP. Top, Schematic structure of COMP constructs used to map those domains (N-terminal, EGF-like, type III, and C-terminal) that bind to BMP-2. Presence or absence of binding between COMP domains and BMP-2 is indicated with + or −, respectively. Bottom, β-Galactosidase activity was used to test the interaction between the C-terminal domain of COMP and BMP-2. Three independent yeast transformants for each pair of plasmids were transferred onto a nitrocellulose membrane, and β-galactosidase activity was determined. E, GST pull-down assay. Purified fusion protein of GST–N terminus, GST-EGF, GST–C terminus, and GST–C type III of COMP were immobilized on GSH-sepharose beads and incubated with purified BMP-2. Proteins trapped by GST fusion protein were examined by immunoblotting with anti–BMP-2 antibodies. Purified BMP-2 (first lane) was used as a positive control. Arrow indicates BMP-2 band. F, Effect of increasing concentrations of unlabeled COMP on the binding of 125I-labeled BMP-2 by VSMC membrane–bound receptors. 125I-labeled BMP-2 was incubated in the absence or presence of unlabeled COMP with VSMC membranes at 4°C for 24 hours. Values are expressed as percentages of specific binding determined in the absence or presence of unlabeled COMP. Each point is the average of triplicate determinations. The fitted curve was obtained by use of GraphPad Prism 5.0. G, COMP retarded the competitive inhibition of unlabeled BMP-2 to 125I-labeled BMP-2 receptor binding. Unlabeled BMP-2 (0.462 nmol/L) competitively inhibited 125I–BMP-2 receptor binding, whereas unlabeled COMP (6.5 nmol/L) significantly abolished the effect. Each point is the average of triplicate determinations. *P<0.05.Show Less
Yaoyao Du, Yue Wang, Li Wang, Bo Liu, Qingyun Tian, Chuan-ju Liu, Tao Zhang, Qingbo Xu, Yi Zhu, Oldberg Ake, Yongfen Qi, Chaochu Tang, Wei Kong, Xian Wang
Circulation Research April 2011, 108 (8) 917-928; DOI: https://doi.org/10.1161/CIRCRESAHA.110.234328
By YaoyaoDu, YueWang, LiWang, BoLiu, QingyunTian, Chuan-juLiu, TaoZhang, QingboXu, YiZhu, OldbergAke, YongfenQi, ChaochuTang, WeiKong and XianWang
COMP is degraded, whereas BMP-2 is increased, in uremic radial arteries of patients with end-stage renal failure (CRF). Representative Western blot an...Show More
COMP is degraded, whereas BMP-2 is increased, in uremic radial arteries of patients with end-stage renal failure (CRF). Representative Western blot analysis and quantification of COMP protein, as well as BMP-2 and Runx2, are shown. CRF: uremic patients who underwent arterial venous fistular operation (n=4 to 6); control: CHD (n=8) patients who underwent coronary artery bypass grafting, without complications of diabetes and chronic kidney disease. Results are means±SEM normalized to that of β-actin. *P<0.05.Show Less
James Shaw, Tong Zhang, Marek Rzeszutek, Natalia Yurkova, Delphine Baetz, James R. Davie, Lorrie A. Kirshenbaum
Circulation Research December 2006, 99 (12) 1347-1354; DOI: https://doi.org/10.1161/01.RES.0000251744.06138.50
By JamesShaw, TongZhang, MarekRzeszutek, NataliaYurkova, DelphineBaetz, James R.Davie and Lorrie A.Kirshenbaum
Figure 1. HDAC inhibition disrupts p65-mediated repression of Bnip3 gene transcription. Panel A, Postnatal ventricular myocytes were transfected with...Show More
Figure 1. HDAC inhibition disrupts p65-mediated repression of Bnip3 gene transcription. Panel A, Postnatal ventricular myocytes were transfected with a Bnip3 luciferase reporter with and without a p65 NF-κB expression vector in the presence and absence of trichostatin A (TSA, 10 nM). Panel B, IKKβ-mediated NF-κB activation represses Bnip3 gene transcription in ventricular myocytes. Postnatal ventricular myocytes were infected with adenoviruses encoding IKKβ (IKKβ) in the presence and absence TSA (10 nM). Endogenous Bnip3 in ventricular myocytes was examined by semiquantitative RT-PCR. Data were normalized to house keeping control gene L32. Control cells (CNTL) were transfected with the eukaryotic expression vector pcDNA3 (panel A) or infected with control adenovirus (panel B). Data are expressed as mean ± S.E. (P<0.05). Experiments were repeated at least n=6 with independent culture conditions using replicates of n=3 for each condition tested, *=statistically different from CNTL; ‡=statistically different from p65; N.S.=not significant.Show Less
Figure 1. HDAC inhibition disrupts p65-mediated repression of Bnip3 gene transcription. Panel A, Postnatal ventricular myocytes were transfected with...Show More
