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Atorvastatin upregulates expression of p16 and inhibits proliferation and migration of VSMCS via altered dna methylation
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Abstracts / Atherosclerosis 263 (2017) e111ee282
PO066. ATORVASTATIN UPREGULATES EXPRESSION OF P16 AND INHIBITS PROLIFERATION AND MIGRATION OF VSMCS VIA ALTERED DNA METHYLATION Boqian Zhu, Chengchun Nanjing, China
Tang. Medical School of Southeast University,
Aim: Atorvastatin experts an inhibitory effect on vascular smooth muscle cells (VSMCs) proliferation and its mechanism remains largely unknown. Previous studies reported that p16, as a well-known tumor suppressor, had an influence on proliferation and migration, but the function of p16 in intimal hyperplasia remains vague. In the present study, we aimed to investigate the function of p16 in VSMCs and its mechanism. Methods: The proliferative and migratory activity of VSMCs were determined by CCK8 and Transwell assay. Protein expression levels were detected by Western Blot and mRNA expression levels were measured by RT-PCR. Methylation-specific PCR was performed to assess methylation of genes. Results: We demonstrated the repression of proliferation and migration of VSMCs treated with atorvastatin. Furthermore, we found that the p16 expression was restored and modulation of the DNA methylation in p16 promoter in VSMCs treated with the atorvastatin and AZA. Overexpressed DNMT1 and silencing of DNMT1 expression by AZA or RNA interference modulating the p16 expression. In addition, we found that MAPKs pathway affected the regulation of p16 by DNMT1 and had an influence on proliferation and migration. Finally, we illustrated that atorvastatin inhibits neointima formation and modulate the p16 expression in balloon catheter-injured rat carotid artery. Conclusions: Our study suggests that atorvastatin inhibits neointima formation and modulate the p16 expression via affecting the DNA methylation in the p16 promoter region. These findings indicate that p16 might be a potential biomarker for the assessment of risk of restenosis, as well as serving as a novel therapeutic target.
PO067. FUNDC1 MEDIATED MITOPHAGY ALLEVIATES RAT AORTIC SMOOTH MUSCLE CELL PROLIFERATION, MIGRATION INDUCED BY ANGIOTENSIN II Xin Wang, Xiqiong Han, Naifeng Liu. Department of Cardiology, Zhongda Hospital of Southeast University Medical School, Nanjing, China Aim: VSMC hyperplasia caused by abnormal proliferation and migration is a major contributor to the development of atherosclerosis, and oxidative stress is a major mechanism of VSMC hyperplasia. Mitophagy is a critical cellular process that selectively targets damaged mitochondria for autophagosomal degradation in response to stress preventing oxidative damage. However, the role of mitophagy in VSMC in response to oxidative stress is unknown. The present study is designed to determine the role of FUNDC1 meditated mitophgy on VSMCs proliferation, migration as well as their underlying mechanisms. Methods: The proliferation of VSMC was determined by CCK8 .The migration of VSMCs was measured by transwell chamber. Mitophagy was measured by laser confocal of VSMC co-loaded with lysotracker red and mitotracker green. Mitophagy associated protein such as FUNDC1,LC3 were detected by westernblot. DCFH-DA was used to detect the intracellular ROS. Alterations of MMP was observed using JC-1. Results: The mitophagy flux increased with the VSMC proliferation and migration induced by AngII. Moreover, AngII increased abundance of FUNDC1 as well as the LC3-II/LC3-I ratio. FUNDC1 silencing impaired mitophagy flux and further aggravated AngII induced proliferation and migration.In addition, overexpression of FUNDC1 inhibited AngII induced proliferation and migration, blocked the cell adhesion molecules and matrix metalloproteinase-9 expression induced by Ang II,furthermore, increased the mitochondrial membrane potential and reduced the intracellular reactive oxygen species production. Conclusions: FUNDC1-mediated mitophagy is involved in the VSMC proliferation and migration induced by Ang II ,and increments FUNDC1-
1meditated mitophagy might be a promising approach to prevent atherosclerosis.
PO068. THE EFFECT OF ADIPOSE TISSUE ADHESIVITY TO THE ENDOTHELIUM
PRODUCTS
ON
MONOCYTE
Sona Cejkova1, Hana Kubatova1, Ivana Kralova Lesna1, Filip Thieme2, Jiri Fronek2, Rudolf Poledne1. 1 Institute for Clinical and Experimental Medicine, Laboratory for Atherosclerosis Research, Prague, Czech Republic; 2 Institute for Clinical and Experimental Medicine, Department of Transplant Surgery, Prague, Czech Republic Aim: Adhesion of monocytes to the endothelium is one of the initial steps in the development of atherosclerosis. Adipose tissue secretes a number of cytokines which may affect the endothelium; in particular, it may stimulate monocyte adhesion and infiltration which initiate and accelerate atherosclerosis development. Methods: Adipose tissue-conditioned media (ATCM) were prepared by culturing visceral adipose tissue obtained from living kidney donors. The effect of ATCM on endothelial cells (ECs) was analyzed by changes in adhesiveness of calcein AM-labelled monocytes to the affected ECs using ATCM and quantified by intensity of fluorescence of the adhered monocytes. Concentrations of cytokines in ATCM were measured by Luminex assay. Results: Increasing concentrations of IL-1b, TNF-a, MCP-1, IL-10, and RANTES (CCL5) gradually increases monocyte adhesivity to ECs (correlation coefficients r ¼ 0.69, p < 0.0001; r¼0.45, p<0.0001; r¼0.45, p<0.0001; r¼0.44, p<0.0001 and r¼0.27; p¼0.003, respectively). No such relationship was found for IL-4, IL-5 and CXCL5. Cytokine concentrations in ATCM were not related to individual atherosclerosis risk factors (age, sex, menopause, and BMI). Conclusions: Adhesion of monocytes to ECs was significantly influenced by IL-1b, TNF-a, MCP-1, IL-10 and RANTES released from adipose tissue. The concentrations of these cytokines in ATCM were not related to individual BMI and other risk factors. Acknowledgements: This project was supported by the Charles University, project GA UK No. 592216 and by the project (Ministry of Health, Czech Republic) for development of research organization 00023001 (Institute for Clinical and Experimental Medicine, Prague, Czech Republic) e institutional support.
PO069. DAMAGING FACTORS ON ENDOTHELIAL CELLS AFFECT CIRCULATING ENDOTHELIAL MICROVESICLE COUNT IN PERIPHERAL BLOOD Zekas Vytautas1, Reda Matuzeviciene1, Dovile Karciauskaite1, Asta Burokiene1, Mantas Radzevicius1, Ausra Mazeikiene1, Neringa Janiulioniene2, Zita Kucinskiene1. 1 Vilnius University, Faculty of Medicine - Departament of Physiology, Biochemistry, Microbiology and Laboratory Medicine., Vilnius, Lithuania; 2 Vilnius University hospital Santariskiu Clinics - Center for laboratory medicine., Vilnius, Lithuania Aim: High numbers of circulating microvesicles of different cell origin have been associated with subclinical atherosclerosis. Microvesicles shed from endothelial cell may participate in pathogenesis of oxidative stress, inflammation, coagulation and angiogenesis. The objective of this study was to test the correlation between known atherosclerosis risk factors and microvesicle count. Methods: We included 66 male individuals in the study aged between 25 and 55 years who were apparently healthy or without any acute clinical condition. Informed consent was obtained from all the subjects and the study protocol was approved by the local ethics committee of Vilnius University. Laboratory tests including concentration of C-reactive protein, glucose, total cholesterol, triglycerides, HDL- and LDL-cholesterol were performed using routine techniques. All samples were labeled with anti CD144-FITC, anti CD105-BV421, anti CD42a-PerCP, anti CD62e-PE, anti CD31-APCy7, anti CD61-APC (BD, San Jose) and tested using BD Fortessa cytometer (BD, San Jose). All events were gated by forward light scatter
Abstracts / Atherosclerosis 263 (2017) e111ee282
PO066. ATORVASTATIN UPREGULATES EXPRESSION OF P16 AND INHIBITS PROLIFERATION AND MIGRATION OF VSMCS VIA ALTERED DNA METHYLATION Boqian Zhu, Chengchun Nanjing, China
Tang. Medical School of Southeast University,
Aim: Atorvastatin experts an inhibitory effect on vascular smooth muscle cells (VSMCs) proliferation and its mechanism remains largely unknown. Previous studies reported that p16, as a well-known tumor suppressor, had an influence on proliferation and migration, but the function of p16 in intimal hyperplasia remains vague. In the present study, we aimed to investigate the function of p16 in VSMCs and its mechanism. Methods: The proliferative and migratory activity of VSMCs were determined by CCK8 and Transwell assay. Protein expression levels were detected by Western Blot and mRNA expression levels were measured by RT-PCR. Methylation-specific PCR was performed to assess methylation of genes. Results: We demonstrated the repression of proliferation and migration of VSMCs treated with atorvastatin. Furthermore, we found that the p16 expression was restored and modulation of the DNA methylation in p16 promoter in VSMCs treated with the atorvastatin and AZA. Overexpressed DNMT1 and silencing of DNMT1 expression by AZA or RNA interference modulating the p16 expression. In addition, we found that MAPKs pathway affected the regulation of p16 by DNMT1 and had an influence on proliferation and migration. Finally, we illustrated that atorvastatin inhibits neointima formation and modulate the p16 expression in balloon catheter-injured rat carotid artery. Conclusions: Our study suggests that atorvastatin inhibits neointima formation and modulate the p16 expression via affecting the DNA methylation in the p16 promoter region. These findings indicate that p16 might be a potential biomarker for the assessment of risk of restenosis, as well as serving as a novel therapeutic target.
PO067. FUNDC1 MEDIATED MITOPHAGY ALLEVIATES RAT AORTIC SMOOTH MUSCLE CELL PROLIFERATION, MIGRATION INDUCED BY ANGIOTENSIN II Xin Wang, Xiqiong Han, Naifeng Liu. Department of Cardiology, Zhongda Hospital of Southeast University Medical School, Nanjing, China Aim: VSMC hyperplasia caused by abnormal proliferation and migration is a major contributor to the development of atherosclerosis, and oxidative stress is a major mechanism of VSMC hyperplasia. Mitophagy is a critical cellular process that selectively targets damaged mitochondria for autophagosomal degradation in response to stress preventing oxidative damage. However, the role of mitophagy in VSMC in response to oxidative stress is unknown. The present study is designed to determine the role of FUNDC1 meditated mitophgy on VSMCs proliferation, migration as well as their underlying mechanisms. Methods: The proliferation of VSMC was determined by CCK8 .The migration of VSMCs was measured by transwell chamber. Mitophagy was measured by laser confocal of VSMC co-loaded with lysotracker red and mitotracker green. Mitophagy associated protein such as FUNDC1,LC3 were detected by westernblot. DCFH-DA was used to detect the intracellular ROS. Alterations of MMP was observed using JC-1. Results: The mitophagy flux increased with the VSMC proliferation and migration induced by AngII. Moreover, AngII increased abundance of FUNDC1 as well as the LC3-II/LC3-I ratio. FUNDC1 silencing impaired mitophagy flux and further aggravated AngII induced proliferation and migration.In addition, overexpression of FUNDC1 inhibited AngII induced proliferation and migration, blocked the cell adhesion molecules and matrix metalloproteinase-9 expression induced by Ang II,furthermore, increased the mitochondrial membrane potential and reduced the intracellular reactive oxygen species production. Conclusions: FUNDC1-mediated mitophagy is involved in the VSMC proliferation and migration induced by Ang II ,and increments FUNDC1-
1meditated mitophagy might be a promising approach to prevent atherosclerosis.
PO068. THE EFFECT OF ADIPOSE TISSUE ADHESIVITY TO THE ENDOTHELIUM
PRODUCTS
ON
MONOCYTE
Sona Cejkova1, Hana Kubatova1, Ivana Kralova Lesna1, Filip Thieme2, Jiri Fronek2, Rudolf Poledne1. 1 Institute for Clinical and Experimental Medicine, Laboratory for Atherosclerosis Research, Prague, Czech Republic; 2 Institute for Clinical and Experimental Medicine, Department of Transplant Surgery, Prague, Czech Republic Aim: Adhesion of monocytes to the endothelium is one of the initial steps in the development of atherosclerosis. Adipose tissue secretes a number of cytokines which may affect the endothelium; in particular, it may stimulate monocyte adhesion and infiltration which initiate and accelerate atherosclerosis development. Methods: Adipose tissue-conditioned media (ATCM) were prepared by culturing visceral adipose tissue obtained from living kidney donors. The effect of ATCM on endothelial cells (ECs) was analyzed by changes in adhesiveness of calcein AM-labelled monocytes to the affected ECs using ATCM and quantified by intensity of fluorescence of the adhered monocytes. Concentrations of cytokines in ATCM were measured by Luminex assay. Results: Increasing concentrations of IL-1b, TNF-a, MCP-1, IL-10, and RANTES (CCL5) gradually increases monocyte adhesivity to ECs (correlation coefficients r ¼ 0.69, p < 0.0001; r¼0.45, p<0.0001; r¼0.45, p<0.0001; r¼0.44, p<0.0001 and r¼0.27; p¼0.003, respectively). No such relationship was found for IL-4, IL-5 and CXCL5. Cytokine concentrations in ATCM were not related to individual atherosclerosis risk factors (age, sex, menopause, and BMI). Conclusions: Adhesion of monocytes to ECs was significantly influenced by IL-1b, TNF-a, MCP-1, IL-10 and RANTES released from adipose tissue. The concentrations of these cytokines in ATCM were not related to individual BMI and other risk factors. Acknowledgements: This project was supported by the Charles University, project GA UK No. 592216 and by the project (Ministry of Health, Czech Republic) for development of research organization 00023001 (Institute for Clinical and Experimental Medicine, Prague, Czech Republic) e institutional support.
PO069. DAMAGING FACTORS ON ENDOTHELIAL CELLS AFFECT CIRCULATING ENDOTHELIAL MICROVESICLE COUNT IN PERIPHERAL BLOOD Zekas Vytautas1, Reda Matuzeviciene1, Dovile Karciauskaite1, Asta Burokiene1, Mantas Radzevicius1, Ausra Mazeikiene1, Neringa Janiulioniene2, Zita Kucinskiene1. 1 Vilnius University, Faculty of Medicine - Departament of Physiology, Biochemistry, Microbiology and Laboratory Medicine., Vilnius, Lithuania; 2 Vilnius University hospital Santariskiu Clinics - Center for laboratory medicine., Vilnius, Lithuania Aim: High numbers of circulating microvesicles of different cell origin have been associated with subclinical atherosclerosis. Microvesicles shed from endothelial cell may participate in pathogenesis of oxidative stress, inflammation, coagulation and angiogenesis. The objective of this study was to test the correlation between known atherosclerosis risk factors and microvesicle count. Methods: We included 66 male individuals in the study aged between 25 and 55 years who were apparently healthy or without any acute clinical condition. Informed consent was obtained from all the subjects and the study protocol was approved by the local ethics committee of Vilnius University. Laboratory tests including concentration of C-reactive protein, glucose, total cholesterol, triglycerides, HDL- and LDL-cholesterol were performed using routine techniques. All samples were labeled with anti CD144-FITC, anti CD105-BV421, anti CD42a-PerCP, anti CD62e-PE, anti CD31-APCy7, anti CD61-APC (BD, San Jose) and tested using BD Fortessa cytometer (BD, San Jose). All events were gated by forward light scatter