title: Conditional knockout of TFPI-1 in VSMCs of mice accelerates atherosclerosis by enhancing AMOT/YAP pathway.
abstract: BACKGROUND
  Tissue factor pathway inhibitor-1 (TFPI-1) has multiple functions and its precise role and molecular mechanism during the development of atherosclerosis are not clear.
  
  
  OBJECTIVE
  To determine the effect and molecular mechanism of TFPI-1 deficiency in vascular smooth muscle cells (VSMCs) in atherosclerosis in the apolipoprotein E knockout (ApoE-/-) mouse.
  
  
  RESULTS
  A mouse model with a conditional knockout of TFPI-1 in VSMCs in an atherosclerosis-prone background (ApoE-/-) was generated. Mice were fed a high fat diet for 18weeks and were then euthanized. Arterial trees and aortas were stained with Sudan IV and were labeled via immunohistochemistry. Cell proliferation and migration of VSMCs in atherosclerotic plaques were assessed. More atherosclerotic lesions and higher levels of proliferation and migration of VSMCs were observed in TFPI-1fl/fl/Sma-Cre+ApoE-/-mice. An interaction between TFPI-1 and angiomotin (AMOT) was identified in human VSMCs by mass spectrometry, immunoprecipitation and co-localization analyses. Signal pathway changes were detected by Western blot analysis, and the expression levels of target genes were determined by real-time PCR. Decreased phosphorylation of AMOT and Yes-associated protein 1 (YAP) in TFPI-1fl/fl/Sma-Cre+ApoE-/-mice resulted in increased expression levels of snail family zinc finger 2 (SLUG) and connective tissue growth factor (CTGF), which are target genes of the Hippo signaling pathway that have been verified as atherosclerosis candidate genes.
  
  
  CONCLUSIONS
  Deficiency in TFPI-1 in the VSMCs of ApoE-/-mice accelerated the development of atherosclerosis by promoting the proliferation and migration of VSMCs which may be caused by the decreased phosphorylation of AMOT and YAP.
  
  
  CONCLUSIONS
  TFPI-1 has been found to has an anticoagulant activity, induce cell apoptosis and prevent cell proliferation. For the first time, we constructed a line of conditional knockout mice in which the TPFI-1 gene is deleted in VSMCs. We found that TFPI-1 deficiency clearly promoted the development of atherosclerosis when these mice were crossed into an ApoE-/-background. One notable feature of atherosclerosis is the proliferation and migration of smooth muscle cells. Previous reports involved TFPI-1 do not completely explain the proliferation and migration of VSMCs because heterozygous TF deficient (TF±) mice bred in an ApoE-/-background did not show diminished atherosclerosis compared to TF+/+mice bred in the same background. Our results first confirmed that TFPI-1 interacts with AMOT, which led to a decrease in the phosphorylation of YAP and further increased the genes expression of the proliferation and migration involved. Our results further confirmed that atherosclerosis was a localized disease.
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 20032, China.
authors.forename: Jiajun
authors.initials: J
authors.lastname: Xiao
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 20032, China.
authors.forename: Kaiyue
authors.initials: K
authors.lastname: Jin
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 20032, China.
authors.forename: Jiping
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 20032, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Ma
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 20032, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 20032, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Jiang
authors.affiliation: Cardiovascular Center, Children's Hospital Affiliated to Fudan University, Shanghai 200032, China.
authors.forename: Huijun
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Cardiovascular Medicine, Huashan Hospital Affiliated to Fudan University, Shanghai 200032, China.
authors.forename: Xinping
authors.initials: X
authors.lastname: Luo
authors.affiliation: Shanghai Research Centre for Model Organisms, Shanghai 201203,China.
authors.forename: Jian
authors.initials: J
authors.lastname: Fei
authors.affiliation: Shanghai Research Centre for Model Organisms, Shanghai 201203,China.
authors.forename: Zhugang
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Institute of Geriatrics, PLA Postgraduate School of Medicine, PLA General Hospital, Beijing 100853, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Yang
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 20032, China; Cardiovascular Center, Children's Hospital Affiliated to Fudan University, Shanghai 200032, China. Electronic address: duanma@fudan.edu.cn.
authors.forename: Duan
authors.initials: D
authors.lastname: Ma
mesh_terms: D000091082:Angiomotins; D000818:Animals; D017209:Apoptosis; D050197:Atherosclerosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D005838:Genotype; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000235:genetics*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: C511047:Amot protein, mouse; D000091082:Angiomotins; D036341:Intercellular Signaling Peptides and Proteins; D008074:Lipoproteins; D008840:Microfilament Proteins; C051928:lipoprotein-associated coagulation inhibitor; D012313:RNA
keywords: AMOT; Atherosclerosis; Signaling pathway; TFPI-1; Vascular smooth muscle cell