Yaoyao Du, Cheng Gao, Ziyi Liu, Li Wang, Bo Liu, Fan He, Tao Zhang, Yue Wang, Xiujie Wang, Mingjiang Xu, Guan-Zheng Luo, Yi Zhu, Qingbo Xu, Xian Wang and Wei Kong
Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;32:2580-2588, originally published October 17, 2012
Yaoyao Du, Cheng Gao, Ziyi Liu, Li Wang, Bo Liu, Fan He, Tao Zhang, Yue Wang, Xiujie Wang, Mingjiang Xu, Guan-Zheng Luo, Yi Zhu, Qingbo Xu, Xian Wang, Wei Kong
Arteriosclerosis, Thrombosis, and Vascular Biology November 2012, 32 (11) 2580-2588; DOI: https://doi.org/10.1161/ATVBAHA.112.300206
A disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7) expression is upregulated in calcifying vascular smooth muscle cells (VSMCs) and arteries. A, Representative Western blot analysis and quantification of ADAMTS-7 protein level in radial arteries of patients. Chronic renal failure (CRF): patients with chronic renal failure and uremia who underwent arterial venous fistular surgery (n=4); control (n=5): patients with chronic heart disease but not diabetes or chronic kidney disease who underwent coronary artery bypass grafting. Data are means±SEM normalized to that of GAPDH. *P<0.05. B, Representative Western blot and quantification of ADAMTS-7 protein level in abdominal aortas of rats with CRF treated with sham operation, calcitriol, 5/6 nephrectomization, or 5/6 nephrectomization plus calcitriol (n=5–7 per group). *P<0.05 vs sham control. C and E, Quantification of relative ADAMTS-7 mRNA level during short- (C) and long-term (E) high-phosphate stimulation in rat VSMCs. Representative Western blot and quantification of ADAMTS-7 protein level during short- (D) and long-term (F) high-phosphate stimulation (2.6 mmol/L) in rat VSMCs. Normalization was to β-actin. Data are means±SEM from 3 independent experiments performed in duplicate. *P<0.05 vs 0 hour or control.
Yaoyao Du, Cheng Gao, Ziyi Liu, Li Wang, Bo Liu, Fan He, Tao Zhang, Yue Wang, Xiujie Wang, Mingjiang Xu, Guan-Zheng Luo, Yi Zhu, Qingbo Xu, Xian Wang, Wei Kong
Arteriosclerosis, Thrombosis, and Vascular Biology November 2012, 32 (11) 2580-2588; DOI: https://doi.org/10.1161/ATVBAHA.112.300206
A disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7) facilitates vascular smooth muscle cell (VSMC) calcification in vitro. A, Western blot analysis of the levels of ADAMTS-7 protein, cartilage oligomeric matrix protein (COMP) degradation, bone morphogenetic protein-2 (BMP-2) protein, and total and phosphorylated Smad1/5/8, runt-related transcription factor 2 (Rnx2) and msh homeobox 2 (Msx2) proteins with adenovirus (Ad)-green fluorescent protein (GFP) or Ad-ADAMTS-7 infection at day 6 after high-phosphate stimulation. Pi indicates high phosphate (2.6 mmol/L). Quantification of calcium deposition (B) and Alizarin red S staining (C) with Ad-GFP or Ad-ADAMTS-7 infection at day 12 after high-phosphate stimulation. D, Western blot analysis with 50 nmol/L scramble or ADAMTS-7 small interfering RNA (siRNA) at day 6 after high-phosphate stimulation. Quantification of calcium deposition (E) and Alizarin red S staining (F) with scramble or ADAMTS-7 siRNA at day 12 after stimulation. Data are means±SEM from 3 independent experiments performed in duplicate. *P<0.05. Reddish/purple staining indicates mineral deposition. Scale bar, 20 μm. G, Quantification of calcium deposition with Ad-GFP, Ad-ADAMTS-7, or Ad-ADAMTS-7 plus Ad-COMP after high-phosphate stimulation.
Yaoyao Du, Cheng Gao, Ziyi Liu, Li Wang, Bo Liu, Fan He, Tao Zhang, Yue Wang, Xiujie Wang, Mingjiang Xu, Guan-Zheng Luo, Yi Zhu, Qingbo Xu, Xian Wang, Wei Kong
Arteriosclerosis, Thrombosis, and Vascular Biology November 2012, 32 (11) 2580-2588; DOI: https://doi.org/10.1161/ATVBAHA.112.300206
A disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7) aggravates aortic ring calcification. A, Western blot analysis of ADAMTS-7 protein level in rat abdominal arteries transfected with adenovirus (Ad)-green fluorescent protein (GFP) or Ad-ADAMTS-7 at day 3 after infection. Rat aortas were then cut into aortic rings and cultured in DMEM or calcifying medium containing 3.8 mmol/L PO43− for 6 days. Quantification of calcium deposition in ADAMTS-7–transfected aortic rings at day 6. B, Western blot verification of ADAMTS-7 knockdown in rat carotid arteries with scramble or ADAMTS-7 small interfering RNA (siRNA) for 3 days. The aortas were cut into aortic rings and cultured in DMEM or calcifying medium for 6 days. Quantification of calcium deposition in aortic rings with ADAMTS-7 knockdown at day 6. Data are means±SEM from 3 independent experiments performed in triplicate. *P<0.05.
Yaoyao Du, Cheng Gao, Ziyi Liu, Li Wang, Bo Liu, Fan He, Tao Zhang, Yue Wang, Xiujie Wang, Mingjiang Xu, Guan-Zheng Luo, Yi Zhu, Qingbo Xu, Xian Wang, Wei Kong
Arteriosclerosis, Thrombosis, and Vascular Biology November 2012, 32 (11) 2580-2588; DOI: https://doi.org/10.1161/ATVBAHA.112.300206
A disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7) is a functional target of miR-29a/b in vascular smooth muscle cells (VSMCs). A, Schematic diagram and alignment of miR-29a/b target region within the 3′ untranslated region (UTR) of ADAMTS-7 in human, rat, mouse, and dog. The highly conserved seed sequences of miR-29a/b complementary to ADAMTS-7 3′ UTR are boxed. B, Luciferase activity of reporter constructs containing the 3′ UTR of ADAMTS-7 (deletion or mutation; with the 6-nt seed sequence deleted or mutated). Quantitative real-time polymerase chain reaction and representative Western blot analysis of VSMCs with ADAMTS-7 expression inhibited by mimic miR-29a/b (C) but upregulated by anti–miR-29a/b (D). β-actin was an internal control. Data are means±SEM from 3 independent experiments performed in duplicate. *P<0.05 compared with control. WT indicates wild type.
Yaoyao Du, Cheng Gao, Ziyi Liu, Li Wang, Bo Liu, Fan He, Tao Zhang, Yue Wang, Xiujie Wang, Mingjiang Xu, Guan-Zheng Luo, Yi Zhu, Qingbo Xu, Xian Wang, Wei Kong
Arteriosclerosis, Thrombosis, and Vascular Biology November 2012, 32 (11) 2580-2588; DOI: https://doi.org/10.1161/ATVBAHA.112.300206
miR-29a/b level is suppressed in calcifying vascular smooth muscle cells (VSMCs) and arteries. A and B, Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of mRNA level of miR-29a/b in VSMCs treated without or with short- (A) and long-term (B) high phosphate. RNU5G was an internal control. Data are means±SEM from 3 independent experiments. *P<0.05 vs 0 hour or control. C, Argonaut 2 (Ago2) pull-down assay. Quantitative RT-PCR analysis of mRNA level of miR-29a/b in Ago2 complex in VSMCs stimulated with high phosphate for various times. *P<0.05 vs 0 hour. D, Quantitative RT-PCR analysis of mRNA level of miR-29a/b in rat abdominal aortas with sham operation, calcitriol, 5/6 nephrectomization, or 5/6 nephrectomization plus calcitriol at the time rats were euthanized (n=5–7 per group). *P<0.05 vs sham. E, Quantification of miR-29a/b level in the radial arteries of patients with chronic renal failure. Chronic renal failure (CRF): uremic patients who underwent arterial venous fistular operation (n=5); control (n=5): patients with coronary heart disease who underwent coronary artery bypass grafting. Data are means±SEM normalized to that of RNU5G. *P<0.05. F, Quantification of miR-29a/b level in VSMCs stimulated with high phosphate and infected with Ad-IκB or Ad-LacZ (50 multiplicity of infection) at 1 hour. *P<0.05.
Yaoyao Du, Cheng Gao, Ziyi Liu, Li Wang, Bo Liu, Fan He, Tao Zhang, Yue Wang, Xiujie Wang, Mingjiang Xu, Guan-Zheng Luo, Yi Zhu, Qingbo Xu, Xian Wang, Wei Kong
Arteriosclerosis, Thrombosis, and Vascular Biology November 2012, 32 (11) 2580-2588; DOI: https://doi.org/10.1161/ATVBAHA.112.300206
miR-29a/b inhibits vascular smooth muscle cell (VSMC) calcification by upregulating a disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7). A and B, Representative Western blot of protein expression and quantification of calcium deposition in CaCl2 and miR-29 mimic- or inhibitor-treated rat carotid arteries at day 7. Rat carotid arteries were periadventially treated with 10 μg control mimic miR or mimic miR-29a/b after CaCl2 treatment. Rat carotid arteries were periadventially treated with control anti-miR or anti–miR-29a/b after CaCl2 treatment. Data are means±SEM from 3 independent experiments. *P<0.05. C, Calcium deposition of VSMCs. VSMCs were transfected with control anti-miR or anti–miR-29a/b, then underwent scramble or ADAMTS-7 small interfering RNA (siRNA) transfection and high-phosphate stimulation for an additional 12 days. Data are means±SEM from 3 independent experiments performed in triplicate. *P<0.05. COMP indicates cartilage oligomeric matrix protein; BMP-2, bone morphogenetic protein-2.
Yaoyao Du, Cheng Gao, Ziyi Liu, Li Wang, Bo Liu, Fan He, Tao Zhang, Yue Wang, Xiujie Wang, Mingjiang Xu, Guan-Zheng Luo, Yi Zhu, Qingbo Xu, Xian Wang and Wei Kong
Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;32:2580-2588, originally published October 17, 2012
