Protection Effects Of High Density Lipoprotein From Atherosclerosis And Mechanisms

Dyslipidemia is one of the most important risk factors in atherosclerosis (AS).The elevation of low density lipoprotein cholesterol (LDL-C) may raise the morbidity and mortality of coronary artery disease (CAD),while high-density lipoprotein cholesterol (HDL-C) may protect people from AS. So there were of great importance in preclinical and clinical medicine to investigate the protection Effects and Mechanisms of High Density Lipoprotein from AS.Fast and high-efficient establishment of AS model is the foundation of doing deep research on human AS disease. As one of the mammals, rat has many similarities with the human beings and easy to be obtained with lower cost. So it is very important to succeed in setting up rat AS model. However, rats have their own anti-atherosclerotic effect, therefore, optimizing methods of rat AS modeling is the key to the completion of this study. In our study, a modified high fat diet feed formula plus vitamin D3 injection and PTC A balloon was used to damage the thoracic and abdominal aorta can succeeded to establish hyperlipidemia models and a rat AS model in the shortest time. Rat models of fatty liver, kidney and pneumonia change were also built simultaneously.In our study, the experiment was performed utilizing the condition of natural serum higher level of HDL and very low LDL in rat. The rats were divided randomly into control group, high-fat-diet-fed group, balloon-injuried group and balloon injuried & high-fat-diet-fed group. The results indicated that the high store of HDL in rats can resist AS induced by LDL-C in limit time and promote the expression of ATP binding cassette transporter A1 in the injured vessels.Apolipoprotein-Ⅰ(ApoA-Ⅰ) is the core part of HDL. In our study, The cultured human acute monocytic leakemia(THP-1) cells were induced into foam cells by exposing to phorbol myristate acetate (PMA) and oxidized low-density lipoprotein(ox-LDL). In vitro experiment showed that ApoA-Ⅰcan promote foam cells reverse cholesterol transport (RCT) and the expression of ABCA1 in foam cells and the effect was time and dose dependent relationship. ApoA-Ⅰwas also found to have the effect of reducing expression of MCP-1, VCAM-1 in foam cell.Part one:Establishment of hyperlipidemia model and the optimization of atherosclerosis model in ratsObjectiveTo explore a better way to establish hyperlipidemia model and atherosclerosis model quickly and successfully in rats.Methods1. Optimization of high-fat diet formula:Eight Rats were fed with high fat diet 7 weeks for establishment of hyperlipidemia models. The other four SD rats were fed with normal diet as control group. Total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and HDL-C and Triglyceride (TG) were tested 1 week pre-procedure and 3,7 week after fed with high fat diet.2. In the AS model group, eight rats were suffer the thoracic and abdominal aorta damage by balloon after high fat diet feeding 1 week.3. The rats in AS model group were intramuscularly injected vitamin D3 (60 million U/kg weight) at the right lower limb before high fat diet feeding, leading to a status of calcium overload.4. Thoracic and abdominal aorta injury were examined with scanning electron microscope and light microscope 1 week after operation. Thoracic aorta of Rats were take for histomorphology test at 2 and 6 weeks after operation. The piece of thoracic aorta was fixed in 4%paraformaldehyde, embeding in paraffin, HE staining. At the same time, take livers, lungs and kidneys in AS model group to check for fat deposition 6 weeks after operation.Results1. After high-fat feeding, the levels of TC, LDL-C, and HDL-C in the AS model group were elevated and showed time-dependent relationship, of which TC and LDL-C was significantly increased compared with the pre-feeding, TC level increased 10 times after feeding 7 week, LDL-C went up to 70 times while the HDL-C and TG were slightly elevated. The levels of HDL-C was not significant higher after high-fat feeding 3 week but significantly higher for 7 weeks compared with the high fat diet pre-feeding (P<0.05).2. one week after thoracic and abdominal aorta injury, endothelial cell damage, intima denuded, the exposure of vascular basement membrane collagen could be found under scanning electron microscope in the AS model group. Scattered platelet adhesion on the surface of collagen can also be seen. Endothelial injury extended approximately 6cm from lcm below the aortic arch to the incision point of the abdominal aorta. Under light microscope, the aorta severely damaged was found and neointimal fibrous plaque formation can also be seen at 2 weeks after operation. Typical atheromatous plaques were presented at 6 weeks after operation.3. The livers, lungs and kidneys of rats in the AS model group are found to have morphological changes after high-fat feeding for 7 weeks. Their surface color changed from red into yellow green. Under light microscope, all there organs were found such changes as fat deposition, fatty degeneration and interstitial capillary congestion.Conclusions1.After feeding for 7 weeks with modified high fat diet formula, the hyperlipidemia model was established successfully. The lipid type of the rats was change from higher HDL-C into highest LDL-C status. The HDL-C/LDL-C ratios gradually decreased which in turn made it possible for the induction of vascular oxidative stress injury by hypercholesterolemia, excite to initiation and development of AS.2. A rat atherosclerosis (AS) model can be established successfully by the use of high-fat feeding, calcium overload and the endothelium effective injury for six weeks.Part two:Effects and Mechanisms of High Density Lipopro-tein on Athrosclerosis of RatsObjectiveTo investigate the effects and mechanisms of high density lipoprotein cholesterol (HDL-C) on atherosclerosis of rats. Methods①SD rats were divided randomly into control group, high-fat-diet-fed group, balloon injured group, and balloon injured& high-fat-diet-fed group.②alloon catheter was used to cause intimae injury of thoracic aorta and abdominal aorta in balloon-injured group and balloon injured& high-fat-diet-fed group. High fat diet was employed to realize hyperlipidemia of rats in high-fat-diet-fed group and balloon injuried& high-fat-diet-fed group.③Total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and HDL-C and Triglyceride (TG) were tested 1 week pre-experiment and 3,7 week after fed with high fat diet.④Histomorphology analysis was done 6 weeks after the operation.⑤Immunohistochemistry dyeing includingα-SM-actin,PCNA,ABCA1,MCP-1 and VCAM-1 of the artery were executed 2 and 6 weeks after the operationResults:1. The blood-fat change obviously after high fat diet. The rate of HDL-C/LDL-C get down from 5.5 to 0.55 and 0.29, LDL-C get up from 0.23±0.07 mmol/L to 2.99±1.08mmol/L and 16.77±2.59mmol/L(P<0.01) at 2 and 6 weeks after procedure. HDL and TG slightly increase after feed. The change of serum lipid was similar between high-fat-diet-fed group and balloon injured& high-fat-diet-fed group.2. After procedure 2 weeks, in balloon injured group and balloon injured& high-fat-diet-fed group, it can be seen that thoracic aortic inner elastic lamina fracture and the media thicker than the control group and neointimal formation. Intimal of thoracic aortic was smooth in high-fat-diet-fed group. After procedure 6 weeks, in balloon injured group and balloon injured& high-fat-diet-fed group neointimal was thicken significantly, the former apparent fibrous plaque while the latter shows a typical AS plaque in neointimal and lumen area of the two groups change small, but in the intimae/media area (%), lumen area stenosis rate (%) no significant difference between two group. In the high fat group, it can be seen that intimal surface less smooth and the adhesion of inflammatory cells in the intimal surface which suggest that hyperlipidemia and intimal calcium overload only can cause oxidative stress injury and inflammatory cell adhesion. But endothelial denudation and vessel wall injury is the key factors caused neointimal formation and vascular remodeling. Based on mechanical damage plus hyperlipidemia oxidative stress injuries, intimal hyperplasia was not found to stimulate the synergy in 6 weeks.3. The expression of PCNA,a-SM-actin,MCP-1,VC AM-1 and ABC A1 were raised in high-fat-diet-fed group, balloon-injured group and balloon injured& high-fat-diet-fed group, especially in the last group 2 weeks after the procedure. The expression of MCP-1,VCAM-1 and ABCA1 in balloon injured& high-fat-diet-fed group were more than in high-fat-diet-fed group and balloon-injured group p<0.05. The expression of MCP-1, VCAM-1 in balloon-injured group more than in high-fat-diet-fed group, but the expression of ABCA1 in balloon-injured group less than in high-fat-diet-fed group. After procedure 6 weeks, the expression of MCP-1,VCAM-1 and ABCA1 in high-fat-diet-fed group, balloon-injured group and balloon injured& high-fat-diet-fed group all decreased obviously. Conclusion:①Although very high level of serum LDL-C, the high store of HDL in rats can resist AS induced by LDL-C in limit time.②. Both of Hyperlipidemia and vascular mechanical injury may show to promote synergistic vascular inflammation, but did not found effects for impaired vascular remodeling and neointimal hyperplasia superimposed.③. It is important that high serum lipid factor promote the expression of ABC A1 of thoracic aorta and the high serum HDL-C level in rats could decrease atherosclerosis by overexpression of ABCA1 for promoting Reverse Cholesterol Transport and and anti-inflammatory action of HDL.Part three:Establishment of a THP-1 macrophage-derived foam cells model by induction of ox-LDLObjectiveTo explore a better way to establish a THP-1 macrophage-derived foam cells model successfully.Methods1. The induction and differentiation of macrophages and foam cells:THP-1 monocytes were maintained growing in suspension in RPMI 1640 culture medium containing 10%FBS at 37℃in 5%CO2 incubator, Cellular quantity doubling time was 48h.The cells were induced to differentiate into macrophages by inoculating into at 6-well plate putting coverslip pre-cultured with 105/ml of cell density and exposing to PMA (50ng/ml) for 48h. Then, the cultured THP-1 cells were induced into foam cells by exposing to ox-LDL (50ug/ml) in RPMI1640 medium for 48h.2. Identification of foam cells:cholesterol content was tested by oil red O staining and oxidase methods, then make conduct qualitative and quantitative analysis of foam cells.3. The expressions of ABCA1,MCP-1,VCAM-1 in macrophages and foam cells were tested by Immunofluorescence method.Results1. The THP-1 cells turned into macrophages after treated with PMA (50ng/ml) for 48h, the cell grown manner varied form suspension aggregation to adherent to base of 6-well plate, the cellular shape changed from the round-shaped into polygonal or spindle, stretching pseudopodia, when the macrophages exposing to ox-LDL (50ug/ml) ater 24-48h, the cellular quantity were increased and become more irregular shape, cytoplasmic lipid droplets by the more visible, even as a large number of lipid droplets within the cytoplasm the nucleus pushed to one side, consistent with morphological features of foam cells.2. It can be seen a large number of red granular lipid droplets in foam cells by oil red O staining, Number of positive cells in induction group was significantly higher than non-induced group p<0.05. The volume of cholesterol in ox-LDL group were higher than in the normal control group(423.17mg/ g protein vs 226.70mg/g protein, p<0.05), also cholesterol ester were more than normal control group. (374mg/g protein vs 62mg/g protein, p<0.05),Cholesterol/ cholesterol ester ratio is more than 60%in induction group, suggesting the formation of foam cells.3. It can be found that after treatment with ox-LDL for 48h, the expression of ABCA1, MCP-1 and VCAM-1 was obviously increased in the foam cells by Immunofluorescence method. These results indicate that after THP-1 derived macrophages transforms into foam cells, the expression of ABCA1 in membrane was significantly increased, and oxidative stress done in foam cells, synthesis of inflammatory cytokines MCP-1 and VCAM-1 increased. Conclusions1. The THP-1 cells Induced differentiation into macrophages after treated with PMA (50ng/ml) for 48h.2. The macrophages taked rich lipid,then differention into foam cells after treatment with ox-LDL(50μg/mL) for 48h.3. The foam cells promot the expression of ABC A1 andMCP-1 andVCAM-1 of inflammation factor.Part Four:Influence of apolipoprotein A-I on reverse cholesterol transport and expression of MCP-1, VCAM-1 and ABCA1 in macrophage derived foam cellsObjectiveTo investigate the effects of ApoAI on reverse cholesterol transport and expression of inflammatory factors in macrophage derived foam cells, it isn't can play a anti-inflammatory role?MethodsThe cultured THP-1 cells were induced into foam cells by exposing to PMA and to ox-LDL. Incubated foam cells by apoA-I in different dose(5ug/m1,10ug/m1,15ug/ml,20ug/ml) for 24h and one dose (lOug/ml) for different time (6h.12h.24h); intracellular lipid droplets change was observed by oil red O staining and total cholesterol, free cholesterol and cholesterol ester content were tested by oxidase. The expressions of MCP-1,VCAM-1 and ABC A1 in macrophages and foam cells were tested by Immunofluorescence method before and after treated with apoA-I (10ug/ml) or ABC A1 antibody for 24 hours.Results1. The THP-1 cells turned into typical foam cells after treated with PMA for 48h, and ox-LDL for 48h.2. ApoA-I can promote reverse cholesterol transport in THP-1 macrophage-derived foam cells and reduce the levels of cholesterol in a dose-dependent and time-dependent manner.3. With the increasing dose of apoA I, the expression of ABCA1 mRNA in THP-1 macrophage-derived foam cells did not change significantly, but the expression of ABCA1 protein increased. After being treated with apoA 1(0μg/ml and 5μg/ml,10μg/ml,15μg/ml,20μg/ml), the positive cells of oil red O staining were (55±4)%,(43±9)%, (33±4)%, (28±1)% and (26±2)%.4. Treatment of ABCA1 antibody could increase the expression of ABCAl protein in THP-1 macrophage-derived foam cells.5. After treatment with ox-LDL(50μg/ml), the expression of MCP-1, VCAM-1 and ABCAl was obviously increased in the foam cells compared with that in the macrophages. Compared with the non-interventional group, the expression of MCP-1 and VCAM-1 in the foam cells was significantly decreased after treatment with apoA-I10mg/L(bothp<0.05), and the expression of ABCA1 was obviously increased(p<0.05); Compared with the non-interventional group the expression of MCP-1 and VCAM-1 were significantly increased after interven with ABCA1 antibody(10 mg/L).Conclusions1. apoA I has anti-arthrosclerosis and anti-inflammatory effect, through increasing the expression of ABCAl and promoting reverse cholesterol transport and decreasing expression of MCP-1 and VCAM-1 protein in foam cells.2. ApoA I-ABCA1 channel may participation in reverse cholesterol transport in foam cells, apoA-I played a key role.