title: Protective Role of Smad6 in Inflammation-Induced Valvular Cell Calcification.
abstract: Calcific aortic vascular and valvular disease (CAVD) is associated with hyperlipidemia, the effects of which occur through chronic inflammation. Evidence suggests that inhibitory small mothers against decapentaplegic (I-Smads; Smad6 and 7) regulate valve embryogenesis and may serve as a mitigating factor in CAVD. However, whether I-Smads regulate inflammation-induced calcific vasculopathy is not clear. Therefore, we investigated the role of I-Smads in atherosclerotic calcification. Results showed that expression of Smad6, but not Smad7, was reduced in aortic and valve tissues of hyperlipidemic compared with normolipemic mice, while expression of tumor necrosis factor alpha (TNF-α) was upregulated. To test whether the effects are in response to inflammatory cytokines, we isolated murine aortic valve leaflets and cultured valvular interstitial cells (mVIC) from the normolipemic mice. By immunochemistry, mVICs were strongly positive for vimentin, weakly positive for smooth muscle α actin, and negative for an endothelial cell marker. TNF-α upregulated alkaline phosphatase (ALP) activity and matrix mineralization in mVICs. By gene expression analysis, TNF-α significantly upregulated bone morphogenetic protein 2 (BMP-2) expression while downregulating Smad6 expression. Smad7 expression was not significantly affected. To further test the role of Smad6 on TNF-α-induced valvular cell calcification, we knocked down Smad6 expression using lentiviral transfection. In cells transfected with Smad6 shRNA, TNF-α further augmented ALP activity, expression of BMP-2, Wnt- and redox-regulated genes, and matrix mineralization compared with the control cells. These findings suggest that TNF-α induces valvular and vascular cell calcification, in part, by specifically reducing the expression of a BMP-2 signaling inhibitor, Smad6.
authors: Department of Medicine, University of California, Los Angeles, California.
authors: Xin
authors: X
authors: Li
authors: Departments of Pediatrics, University of California, Los Angeles, California.
authors: Jina
authors: J
authors: Lim
authors: Department of Physiology, University of California, Los Angeles, California.
authors: Jinxiu
authors: J
authors: Lu
authors: Department of Medicine, University of California, Los Angeles, California.
authors: Taylor M
authors: TM
authors: Pedego
authors: Department of Medicine, University of California, Los Angeles, California.|Department of Physiology, University of California, Los Angeles, California.|Department of Bioengineering, University of California, Los Angeles, California.
authors: Linda
authors: L
authors: Demer
authors: Department of Medicine, University of California, Los Angeles, California.
authors: Yin
authors: Y
authors: Tintut
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D001011:Aorta / Q000378:metabolism / Q000473:pathology; D001021:Aortic Valve / Q000378:metabolism / Q000473:pathology*; D001024:Aortic Valve Stenosis / Q000235:genetics*; D055396:Bone Morphogenetic Protein 2 / Q000096:biosynthesis*; D002114:Calcinosis / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007249:Inflammation / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000235:genetics; D051905:Smad6 Protein / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D051905:Smad6 Protein; C497803:Smad6 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D000469:Alkaline Phosphatase
keywords: BONE MORPHOGENETIC PROTEIN-2; CALCIFICATION; TNF-α; TUMOR NECROSIS FACTOR-α; VALVULAR CELLS