Jiajun Xiao1, Kaiyue Jin1, Jiping Wang1, Jing Ma1, Jin Zhang1, Nan Jiang1, Huijun Wang2, Xinping Luo3, Jian Fei4, Zhugang Wang4, Xiao Yang5, Duan Ma6. 1. 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. 2. Cardiovascular Center, Children's Hospital Affiliated to Fudan University, Shanghai 200032, China. 3. Department of Cardiovascular Medicine, Huashan Hospital Affiliated to Fudan University, Shanghai 200032, China. 4. Shanghai Research Centre for Model Organisms, Shanghai 201203,China. 5. Institute of Geriatrics, PLA Postgraduate School of Medicine, PLA General Hospital, Beijing 100853, China. 6. 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.
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. OBJECTIVES: 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. METHODS AND 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. CONCLUSION: 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. SIGNIFICANCE: 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.
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. OBJECTIVES: 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. METHODS AND 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. CONCLUSION: 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. SIGNIFICANCE: 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: Tingting Huang; Yuhang Zhou; Jinglin Zhang; Alfred S L Cheng; Jun Yu; Ka Fai To; Wei Kang Journal: J Transl Med Date: 2018-04-12 Impact factor: 5.531