Twist-related protein 1 negatively regulated osteoblastic transdifferentiation of human aortic valve interstitial cells by directly inhibiting runt-related.

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Twist-related protein 1 negatively regulated osteoblastic transdifferentiation of human aortic valve interstitial cells by directly inhibiting runt-related transcription factor 2 Xi-Wu Zhang, MD, Bo-Yao Zhang, MD, Shu-Wei Wang, MD, De-Jun Gong, MD, Lin Han, MD, Zhi-Yun Xu, MD, Xiao-Hong Liu, MD The Journal of Thoracic and Cardiovascular Surgery Volume 148, Issue 4, Pages 1700-1708.e1 (October 2014) DOI: 10.1016/j.jtcvs.2014.02.084 Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 The expression of twist-related protein 1 (TWIST1) in human calcified and noncalcified aortic valves. A, Quantitative real-time polymerase chain reaction showed that TWIST1 mRNA levels were lower in calcified valves (calcific aortic valve disease [CAVD], n = 28) than in noncalcified valves (control [CTL], n = 12). *P < .05 versus CTL. B, Western blot showed that TWIST1 protein expression was also downregulated in the calcified valves (CAVD). β-ACTIN was used as a control for protein loading. C-F, Immunohistochemical staining for TWIST1 and runt-related transcription factor 2 (RUNX2) in calcified (CAVD) and normal (CTL) aortic valves (×400), with insets of greater magnification. Arrows point to positive staining. f, Fibrosa; s, spongiosa; v, ventricularis; mRNA, messenger RNA. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 1700-1708.e1DOI: (10.1016/j.jtcvs.2014.02.084) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Establishment of human aortic valve interstitial cell (VIC) osteogenic models with osteogenesis-inducing medium (OIM). A-C, Quantitative real-time polymerase chain reaction showed that mRNA levels of the osteogenic markers were significantly upregulated in VICs treated with OIM for 2 (OIM2) or 4 (OIM4) days. Data represented as the fold change. D, The protein levels of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) increased in VICs treated with OIM. E, Alkaline phosphatase (ALP) activity also increased. The data were normalized to the total protein level. F and G, TWIST1 expression was also upregulated during VIC osteoblastic transdifferentiation in vitro (n = 3 experiments for all tests; error bars represent ± standard deviation). *P < .05 versus control (CTL). GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; TWIST1, twist-related protein 1. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 1700-1708.e1DOI: (10.1016/j.jtcvs.2014.02.084) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Overexpression of twist-related protein 1 (TWIST1) inhibited the expression of runt-related transcription factor 2 (RUNX2) and its downstream osteogenic markers in valve interstitial cell (VIC) osteogenic models. VICs were transfected with TWIST1 expression plasmid (pTWIST1) and treated with osteogenesis-inducing medium (OIM) for 4 days. The cells transfected with an empty vector (negative control empty vector plasmid [pNC]) served as the negative control. A, The VICs cultured in vitro showed elongated or spindle-shaped morphology. Smaller cell clusters formed in the (C) pTWIST1 group than in the (B) pNC group. The overexpression of TWIST1 was verified through (D) quantitative real-time polymerase chain reaction and (E) Western blot. F, The expression of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), type I collagen (COL1), and alkaline phosphatase (ALP) mRNA was downregulated in the pTWIST1 group. G, The expression of RUNX2, OCN, and osteopontin (OPN) was inhibited by TWIST1 in VIC osteogenic models. H, ALP activity also decreased in the VIC osteogenic models transfected with pTWIST1 (n = 3 experiments for all tests; error bars represent ± standard deviation). *P < .05 versus pNC; #P < .05 versus pNC plus OIM. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 1700-1708.e1DOI: (10.1016/j.jtcvs.2014.02.084) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Downregulation of twist-related protein 1 (TWIST1) increased the mRNA expression of runt-related transcription factor 2 (RUNX2) and its downstream osteogenic markers. Valve interstitial cell (VIC) osteogenic models were transfected with TWIST1-specific small interfering RNA (si-TWIST1) or scrambled negative control siRNA (si-NC). A and B, A carboxyfluorescein-siRNA was used to test the transfection efficiency, which could reach ≤90%. C, TWIST1 mRNA expression was decreased by 68%. D-G, The mRNA expression level of RUNX2, osteocalcin (OCN), type I collagen (COL1), and alkaline phosphatase (ALP) in the si-TWIST1 group was upregulated by 49%, 53%, 93%, and 49%, respectively (n = 3 experiments for all tests; error bars represent ± standard deviation). *P < .05 versus si-NC. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 1700-1708.e1DOI: (10.1016/j.jtcvs.2014.02.084) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Twist-related protein 1 (TWIST1) suppressed runt-related transcription factor 2 (RUNX2) expression by directly binding to the RUNX2 P2 promoter. A, Nine E-boxes (CANNTG) were in the RUNX2 P2 promoter region. B and C, Two putative target E-boxes of TWIST1 were identified using chromatin immunoprecipitation (ChIP) assays, E-box-1333 and E-box-820. We cloned the 1466-bp region that included these 2 E-boxes into a pGL3-basic vector (A) and constructed 2 plasmids that were mutated in E-box-1333 or E-box-820 using site-directed mutagenesis. D, The dual luciferase reporter system showed that TWIST1 could inhibit the promoter activity of pGL3-RUNX2-P2 and pGL3-P2-1333M but had little repression effect on pGL3-P2-820M (n = 3 experiments for all tests; error bars represent ± standard deviation). *P < .05; ΔP > .05. IgG, Immunoglobulin G. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 1700-1708.e1DOI: (10.1016/j.jtcvs.2014.02.084) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions

Figure E1 Vimentin and CD31 detection in valve interstitial cells (VICs). A, Vimentin (a mesenchymal cell marker) was expressed in all the cells (green fluorescence). B, Flow cytometry showed that the CD31-positive rate was <2%. FSC-A, Forward scatter area; APC-A, allophycocyanin area; FL6-A, fluorescence 6 (CD31 herein) area. The Journal of Thoracic and Cardiovascular Surgery 2014 148, 1700-1708.e1DOI: (10.1016/j.jtcvs.2014.02.084) Copyright © 2014 The American Association for Thoracic Surgery Terms and Conditions