The Effects And Mechanisms Of Endothelial Metrnl On Atherosclerosis

Atherosclerosis is a choronic inflammatory disease with complex etiologies,resulting in malignant cardio-cerebro-vascular events.The effectiveness of traditional treatments is limited.It is necessary for us to look for new targets.Vascular endothelial cells play an important role in maintaining vascular homeostasis,and dysfunction of endothelial cells is widely considered as the initial factor of atherosclerosis.Vascular endothelial cells are highly active endocrine organ.The research on synthesis and secretion of proteins in endothelial cells is of great importance to elucidate the pathogenesis of atherosclerosis.Metrnl is a new adipokine identified by our laboratory with caloric restriction model and bioinformatic methods.Early research in our lab suggested Metrnl was highly expressed in the vascular endothelial cells.Metrnl can be secreted from endothelial cells,and endothelial cells are the main source of Metrnl in circulation,so Metrnl may be an active substance required for maintaining vascular homeostasis.This project is to investigate the effects and mechanisms of endothelial Metrnl in the high-fat induced and spontaneous atherosclerosis.We try to provide evidence for Metrnl protein to be a new target in the prevention and treatment of atherosclerosis.Objectives1.To investigate the effects of atherosclerotic high-fat diet（HFD）on the basic physiological indexes of mice;2.To investigate the role of endothelial Metrnl in the development of high-fat induced and spontaneous atherosclerosis;3.To investigate the mechanisms of endothelial Metrnl in atherosclerosis.Methods1.C57BL/6J and Apo E^-/-mice were fed with atherosclerotic HFD to investigate its effects on body weight,glucose,and lipid levels,and to observe the effect of fasting time on the results of blood lipids testing.2.EC-Metrnl^-/-mice have been constructed previously and were further crossed with Apo E^-/-mice to obtain Apo E^-/-EC-Metrnl^-/-and the control mice Apo E^-/-WT.The expression of Metrnl in aorta and other tissues and the concentration of Metrnl protein in serum were detected and compared between Apo E^-/-EC-Metrnl^-/-and the control mice.3.The atherosclerosis of Apo E^-/-EC-Metrnl^-/-and control mice under different conditions was investigated:（1）mice aged 2-3 or 7-9 months were fed with HFD for 10weeks to investigate the role of endothelial Metrnl in the high-fat induced atherosclerosis.（2）Mice were fed with normal diet for 1 year or more to investigate the role of endothelial Metrnl in the development of spontaneous atherosclerosis.4.The inflammatory infiltration of aortic root in Apo E^-/-EC-Metrnl^-/-and control mice was investigated by F4/80 and IL-6 immunohistochemistry.5.In vitro,the effects of Metrnl deficiency on the inflammation in human umbilical vein endothelial cells（HUVECs）under normal and oxidized low-density lipoprotein（ox-LDL）stimulated conditions were investigated.6.The expressions of Metrnl in aorta and bone marrow were verified by m RNA and protein levels in C57BL/6J mice,and the efficiency of Metrnl knockout of bone marrow in EC-Metrnl^-/-mice was also investigated.The atherosclerosis mouse model with bone marrow Metrnl specific knockout was established by bone marrow transplantation（BMT）.The details were as follows:Apo E^-/-EC-Metrnl^-/-mice and control mice Apo E^-/-WT were donors,and Apo E^-/-mice were receptors.Apo E^-/-mice received radiation exposure to damage bone marrow totally,and then received bone marrow cells from donors.After 1 month of recovery,mice were fed with HFD for 10 weeks to investigate atherosclerotic plaques.Results1.Body weight and blood glucose,cholesterol,low density lipoprotein cholesterol （LDL-c）and high density lipoprotein cholesterol（HDL-c）levels were significantly increased in the mice fed with HFD,while the level of TG was decreased.Compared with no fasting group,the TG level was significantly decreased in the mice fasting for 12 h.2.The expression of Metrnl m RNA in aorta of Apo E^-/-EC-Metrnl^-/-mice was significantly decreased,but was unchanged in other tissues.In addition,the serum level of Metrnl was decreased significantly.The results were consistent with the previous study in EC-Metrnl^-/-mice.3.Both Apo E^-/-EC-Metrnl^-/-and control mice aged 2-3 months developed atherosclerotic plaques after HFD for 10 weeks.The areas of aortic plaques,and necrotic injuries and lipid accumulations of aortic root in Apo E^-/-EC-metrnl^-/-mice were significantly heavier than those in control mice.There were no significant differences in body weight,food intake,blood glucose and blood lipids between the two groups.Similarly,in the case of 7-9-month-old mice fed with HFD for 10 weeks and spontaneous atherosclerosis（normal diet for more than 12 months）,atherosclerotic plaques were also observed in both groups,with more severe atherosclerosis in Apo E^-/-EC-Metrnl^-/-mice than control mice.4.The immunohistochemical staining of F4/80 and IL-6 in the aortic root of Apo E^-/-EC-Metrnl^-/-mice was more obvious than control mice,suggesting that the inflammatory infiltration of the aorta in this group was aggravated.5.In vitro,experiments showed that the expression levels of pro-inflammatory factors IL-6,IL-1βand MCP-1 were significantly increased in HUVECs with Metrnl knockdown under normal conditions.Furthermore,the expressions of pro-inflammatory factors IL-6,TNF-α,and IL-1βwere increased under ox-LDL stimulation.6.The expression of Metrnl in bone marrow was much lower than that in aorta in normal mice,and it was partly knocked down in the bone marrow of EC-Metrnl^-/-mice.Mice developed atherosclerotic plaques fed with HFD for 10 weeks.There were no significant differences in atherosclerotic plaques between Apo E^-/-mice receiving bone marrow donor cells from Apo E^-/-EC-Metrnl^-/-mice and control mice(Apo E^-/-WT mice).ConclusionEndothelial Metrnl deficiency can promote the development of high-fat induced and spontaneous AS.The underlying mechanism is at least related to cellular inflammation induced by Metrnl deficiency in endothelial cells.Metrnl may be a new target for the prevention and treatment of AS.In addition,mice fed with HFD are characterized by increase in TC and LDL-c levels and decrease in TG levels.The fasting time for 12 hours can affect the serum TG level,with no changes in other serum lipids levels.