Study On The Roles And Molecular Mechanism Of FGF21 Involving On The Pathogenesis Of Atherosclerosis

Background:Coronary atherosclerotic heart disease is a heart disease involving in lumen of blood vessel narrowing or blocking by coronary atherosclerosis, the alteration of coronary artery function induced by myocardial ischemia anoxemia or necrosis, generally called coronary heart disease (CHD). It is about 1/3-1/2 death toll who die caused by CHD in western country, and in our country, the morbidity of CHD was increased strikingly, and the basal population is so big one in country, so how to regulate and manage the development of this disease is becoming a urgent problem now. Atherosclerosis (AS), characteristiced by artery vessel wall increased thickness, losing elasticity or lumens narrowing, is an important pathology basis in cardiovascular and cerebrovascular diseases, also a key pivot in metabolism syndrome. Epidemiology and clinical research showed that AS was mainly happened in middle-age population above 40 years old, and developed sharply after 49 years old, however it was found early atherosclerotic pathological changes in young adults even in children necropsy. CHD, the result of the development of AS, is a common disease which seriously harm to human health. However, even though statin drugs showed a significant resistance to the pathological development of AS in clinical trials, it can not inhibit and reverse deterioration leision in AS patients. So, it is very important to explore the exact molecular mechanism of AS pathogenesis, confirm the new reaction targets of drug and develop an effective new drug to resist the onset and development of atherosclerosis for relieving the patient's pain and social burden.FGF21 is a polypeptide including 210 amino acid residues, predominantly expressed in liver and adipose tissue. Recently studies showed that, under the physiological and pathological conditions, FGF21 was directly involved in the regulation of lipid and glucose metabolism, and closely associated with a series of metabolic syndrome caused by lipid and glucose metabolic disorder. In addition, animal and clinical research revealed that FGF21 was closely related to the morbidity of many metabolism syndrome caused by various kinds of obesity, and more importantly, the treatment of FGF21 were significantly deceased the level of risk factor of cardiovascular diseases in vitro. However, no data was found to focus the relationship between FGF21 and coronary heart disease in animal or clinical research. Lipid metabolic disorder is one of important mechanisms in arteriosclerosis, and hyperglycemia is a major high risk factor in the pathogenesis of AS. However, whether FGF21 is related to the onset and development of AS? What types of pathways to regulate the procedure of metabolism in vitro? All of these questions require further investigation.Objective:The aim in this project is to explore on the pathogenesis of AS, on basis of relevant experiments including the manifestation of FGF21 in CHD patients and apolipoprotein E knockout (apoEKO) animals, recombinant FGF21 interrupting the pathogenesis of atherosclerosis in apoEKO mice and cellular molecular mechanism in vitro, we were widely explore and reveal the role and exact molecular mechanism of FGF21 in the pathogenesis of AS.Methods:1. Based on the clinical diagnostic criteria of CHD published by WHO in 1999, 135 CHD patients and age and gender-matched 61 normal control people were recruited in this study. Serum levels of FGF21 were determined by enzyme-linked immunosorbent assay (ELISA) in CHD patients and control subjects, meanwhile, serum circulation levels of FGF21 related to other clinical and biochemical indicator, mass index and risk factors were analyzed statistically.2.8 weeks old apoE KO mice were involved in this study, and fed by high fat diet to induce atherosclerosis for 4 weeks, serum samples were collected and measured the level of serum FGF21 concentration by ELISA assay. The expressions of FGF21 in artery vascular tissue, ascending aorta and liver tissue were examined by Real-time quantitative PCR and immunohistochemistry techniques. Simultaneously, when apoE KO mice were induced succeffully to the formation of atherosclerosis fed by high fat diet, pravastatins were given by drinking water in a dose of 40mg/kg-d in 12 weeks old. Serum sample of apoE KO mice in 12 and 24 weeks were collected and used to examine serum FGF21 levels by ELISA assay, and the expression of FGF21 in vascular tissue and liver tissue were detected by Real-time quantitative PCR.3.8 weeks old apoE KO mice were involved in this study, and treated with recombinant FGF21 in 100 or 400μg/kg dose by subcutaneous injection respectively, at the same time, another group mice were injected physiological saline as a control. Body weight were measured weekly during FGF21 treatment, the change of body weight will be observed. Mice will be sacrificed after 6 weeks of administration of FGF21, serum sample will be collected, and lipid profiles were tested. Aorta, arcus aortae and liver were collected. The effect caused by FGF21 in the vascular disease and liver injury in apoE KO mice were estimated in histomorphology and molecular levels by western blot, QPCR, immunohistochemisty methods and so on.4. Make use of the cellular model to observe the physiological characteristics of HUVEC and macrophage as well as its relative signaling pathway mediated by FGF21.(1) To reveal the expression of FGF21 in HUVEC and the effect caused by oxLDL; (2) To explore the effect of the expression of PON1 caused by exogenous FGF21 in HUVEC; (3) To observe the effect of the accumulation of lipid in THP-1-derivated macrophages mediated by FGF21; (4) To investigate the change of relative signaling pathway mediated by FGF21 in HUVEC and THP-1-derivated Macrophages.Results:1. Circulating serum FGF21 levels are increased in CHD patients(1) Median serum FGF21 levels were significantly higher in CHD patients (513.5±47.5ng/l) than that of normal control subjects (167.9±15.6 ng/1) (P< 0.0001).(2) Serum FGF21 levels in CHD patients with diabetes, hypertension, or both were higher than that of patients without these comorbidities(P<0.05).(3) Serum level of FGF21 correlated positively with triglycerides, fasting blood glucose, apolipoprotein B100, insulin and HOMA-IR, but negatively with HDL-C and apolipoprotein Al after adjusting for BMI.(4) Logistic regression analysis demonstrated that FGF21 showed an independent association with triglyceride and apolipoprotein Al. 2. The expression levels of FGF21 are related to the change of pathology of arteriosclerosis in apoE KO mice.(1) The expression levels of FGF21 in apoE KO mice increased markedly with the pathological change of AS.①Circulating serum FGF21 levels in 8 weeks old apoE KO mice were low, and its were significantly increased with the development of atherosclerosis.②Compared with the same genetic background of C57BL/6j mouse, FGF21 mRNA expression levels were significant high in the liver, aorta, kidney, spleen in the 24 weeks old apoEKO mice. Compared with other tissues, liver and aortic tissue showed a higher level in FGF21 expression.③In 24-week old apoEKO mice, the expression of FGF21 mRNA level were significantly higher in the vascular tissue than that of the same genetic background and age wild-type C57BL/6j mouse, and western blotting analysis got a similar result. Immunohistochemisty results showed that FGF21 has a higher expression in liver tissue than that of wild type mice.(2) Pravastatin decreased the expression levels of FGF21 in apoE KO mice①Serum FGF21 levels in 12-week old apoE KO mice markedly increased compared with those in 8-week old apoE KO mice, and after treatment with pravastatin for 12 weeks in 40mg/kg-d dose, serum FGF21 concentration significantly decreased compared with those in 12-week old (P<0.05).②Compared with the positive control group (HFD group), FGF21 mRNA expression significantly decreased in liver tissue (P<0.05), and an increase tendency of FGF21 mRNA expressive levels were found in the kidney tissue after treatment pravastatin, but no statistic significant change was found between pravastatin and HFD group.③Compared with the control group, PON1 expression levels in aortic tissue were markedly increased in apoE KO mice, however, it was down-regulated after treatment with paravastain.3. Recombinant FGF21 relieved the pathogenesis of atherosclerosis in apoE KO mice(1) FGF21 treatment directly affected body weight index in apoEKO mouse: FGF21 attenuated the increasing of body weight, decreased the weight of whole liver tissue, and also decreased notably the ratio of liver/body weight in apoEKO mice and presented a dose-dependent effect.(2) FGF21 significantly improved abnormal lipid profiles in apoEKO mouse: FGF21 treated had down-regulated serum triglyceride, LDL, FFA levels, and up-regulated the serum level of HDL in apoEKO mice.(3) FGF21 directly affect the expression of aim genes involving in hepatic metabolism in apoE KO mouse:①FGF21 significantly alleviated the accumulation of triglyceride and free fatty acid in liver tissue;②FGF21 treatment showed accelerated hepatic lipid oxidation, suppressed lipogenesis, cholesterol and bile acid metabolism in apoE KO mouse;③FGF21 treatment released liver tissue injury which was induced by HFD and lipid metabolic disorder;④the accumulation of hepatic glycogen was predominantly alleviated by FGF21 treatment.⑤FGF21 treatment significantly down-regulated the expression of p-p38 or p-JNK signaling pathway molecules in apoE KO mice.(4) Compared with control group, the expression level of ABCA1 and ABCG1 mRNA in small intestine were notably up-regulated by FGF21 treatment.(5) FGF21 treatment can alleviate the pathogenesis of atherosclerosis:①FGF21 can inhibit aortic intimal thickening in apoE KO mice;②FGF21 resisted the development of atherosclerotic plaque lesion in apoEKO mice;③FGF21 decreased the expression of ICAM-1 in aortic intima.4. Study focusing on FGF21, HUVEC and macrophages in vitro(1) The expression levels of FGF21 mRNA in HUVEC were increased dependently on the concentration of oxLDL, and depended on the time of oxLDL treatment.(2) FGF21 up-regulated PON1 expression in cultured HUVEC presented a dose and time dependencies and related to ERK1/2 signaling pathway.(3) FGF21 can down-regulated the activities of Caspase-3 and caspase-8 to inhibit the apoptosis induced by paclitaxel.(4) FGF21 deceased the accumulation of lipid in THP-1-derivated macrophages by promote lipid efflux.(5) HUVEC treated with FGF21 for 20min could activate 13 kinds of phosphorylation proteins including p38α,ERK1/2,JNK,GSK-3α/βand so on; and FGF21 could activate about 7 kinds of phosphorylation proteins including p38a, Paxillin, Akt, Lyn and so on in THP-1 derivated macrophages.Conclusion:1. Serum levels of FGF-21 are increased in coronary heart disease patients and are independently associated with adverse lipid profile.2. FGF21 expression were significantly increased in apoE KO mice model, and highly expressed in the liver and blood vessels tissue. Pravastatin directly affected the expression levels of FGF21 in apoE KO mice model fed by high fat diet.3. Gaining of body weight and relevant pathological changes induced by apoE-deficiency and high fat diet were significantly alleviated by recombinant FGF21 treatment in apoE KO mouse.4. HUVEC is one type of cells that can express FGF21, FGF21 up-regulated PON1 expression in cultured HUVEC were mediated by ERK1/2 signaling pathway, and FGF21 can attenuate apoptosis induced by paclitaxel. FGF21 deceased the accumulation of lipid by promote lipid efflux on basis of the expression of ABCA1 genes. FGF21-treatment induced a serial of phosphrylation reaction in signaling pathway molecular in HUVEC.