| Backgroud and objectivesInvestigations in recent years have demonstrated that endothelial cells (VEC) lesion and endothelial dysfunction is the initiating and early event in the development of atherosclerosis (AS). At the same time, as more is learned about the natural history of the development of AS, it is clear that the injury to the integrity and function of VEC begins in childhood and AS is a pathological process progressed slowly but steadily, which begins in the adolescent and becomes symptomatic during the adults. The target organs may appeare some reversible pathological changes in childhood, especially for these children with high risk fators for cardiovascular disease, such as atherosclerotic family history, simple obesity and diabetes mellitus. Therefore, it is crucial to detect and prevent vascular endothelial dysfunction earlier for the prevention of AS.Hypercholesterolemia (HC) is an independent risk factor for the development and progression of AS. More and more studies have indicated that the oxidized low-density lipoprotein (ox-LDL) plays an important role in causing endothelial dysfunction and initiating AS. The uptake of low density lipoprotein (LDL),especially of ox-LDL by endothelial cells is a key step in the pathogenesis of AS. The captured ox-LDL activates a variety of intracellular processes that induce the expression of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) and vascular intercellular adhesion molecule-1 (VCAM-1) and chemoattactant factors like monocyte chemoattractant protein 1 (MCP-1). The expression of adhesion molecules by endothelial cells and the local production of chemoattractants are key molecular events for monocyte migration/accumulation, while the migration and accumulation of monocytes into the subendothelial space of the vessel wall is one of the earliest detectable responses in the formation of atherosclerotic lesions. Down-regulation of such chemoattractant mediators and the prevention of tissue macrophage accumulation are recognized as important mechanisms of drugs targeting atherogenesis. Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) was initially identified as the major receptor for ox-LDL in endothelial cells, which allows the uptake of ox-LDL into endothelial cells. Then its inducible expression was also observed in macrophages and smooth muscle cells (SMCs) and might play an important role in the uptake of ox-LDL and the subsequent formation of foam cells by macrophages and SMCs, which indicated that the expression of LOX-1 is important both in the initiating link and in the developping process of AS. So, now LOX-1 is recognized as the major receptor for ox-LDL, plays a critical role in the develepment of endothelial dysfunction and AS. Identification of soluble LOX-1 in vivo may provide a novel diagnostic tool for the evaluation and prediction of AS and vascular disease and the regulation of this receptor and understanding of its signal transduction pathways may lead to new therapies in disease states characterized by endothelial dysfunction.Clinical data and experimental studies have established the important role of abnormal lipid metabolism, especially the elevated plasma concentrations of LDL cholesterol in the causation of AS and so enthroned the 3-hydroxy-3-metylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) as a mainstay in themanagement of patients with HC and AS. Clinical data have shown that statins could significantly reduce cardiovascular mortality and morbidity. At the same time, emerging experimental data underline the role of vascular renin-angiotensin systems (RAS) in mediating the early stages of vascular endothelial dysfunction and it is involved in the pathogenesis of AS. Hypertension (HT) and HC are both independent risk factors for the development of AS and there seems a strong synergy action between them in terms of atherogenesis. Angiontensin II (Angll) expression and activity have been demonstrated by immunohistochemistry increased dramatically in patients with AS and experimental animal models of HC. Moreover, multiple randomized controlled clinical trials show beneficial clinical outcomes, including retarding AS and cardioprotection, with the use of angiotensin converting enzyme inhibitors (ACEI) and angiotensin II 1 receptor (AT1R) blockers (ATlRBs). But the certain mechanism of RAS and Angll involved in the atherogenesis is still unclear. In recent years it is found that Angll can regulate the expression of LOX-1 by VECs in transcriptive level. Co-incubation of human coronary artery endothelial cells (HCAECs) with Ang II markedly increased LOX-1 mRNA and protein expression and the increase of LOX-1 expression was dependent on Ang II concentration. Moreover, these effects of Ang II were completely blocked by pretreatment of HCAECs with losartan, a specific AT1RB. Ang II caused a concentration-dependent increase in ox-LDL uptake by HCAECs and so induced a decrease in VEC viability. These observations indicated that Ang II enhances ox-LDL-mediated injury to HCAECs through upregulating LOX-1 expression, and the upregulating effects of Ang II are mediated by AT1R activation. But these initial investations were performed in vitro.In vivo, there are few investations and reports on whether HC and Angll have regulating effects on the expression of LOX-1 in VEC and whether hypolipidemic drugs of statins and ATlRBs losartan could prevent its expression. In this study, we established hypercholesterolemic andatherosclerotic model in rabbits by high-cholesterol diet and/or exogenous injection of Angll to observe the effect of HC and Angll on the expression of LOX-1 and its relationship with atherogenesis. At the same time the present study was designed to investigate the prevention effect of statins and losartan on the atherosclerotic lesion, the vascular inflammation and the protein and gene expressing level of LOX-1 in the vascular wall and intended to explore the effective intervention methods to endothelial disfunction and early atherosclerotic changes. Methods:1. The establishment of atherosclerotic rabbit model1.1 The atherosclerotic rabbit model induced by high-cholesterol diet: 32 male New Zealand white rabbits were assigned randomly to one of the four dietary regimens for 16 weeks: (1) Control group, rabbits fed with standard chow, n=8; (2) HC-diet group, rabbits fed with high-cholesterol diet (HC-diet, containing 1% cholesterol and 5% peanut oil ), n=8; (3) Statin group, rabbits fed with HC-diet plus fluvastatin 10 mg *kg~' *d~', gavage, n= 8. (4) Losartan group, rabbits fed with HC-diet plus losartan 25 mg ? kg"' ? d~', gavage, n= 8.1.2 The atherosclerotic rabbit model induced by HC-diet plus exogenous supply of Angll: 32 male New Zealand white rabbits were assigned randomly to one of the four regimens: (1) Normal control group, rabbits fed with normal chow plus injection of normal saline (NS), n^S; (2) Normal injection group, rabbits fed with normal chow plus injection of Angll, n=8; (3) HC-control group, rabbits fed with HC-diet plus injection of NS, n=8; (4) HC-injection group, rabbits fed with HC-diet plus injection of Angll, n=8. High cholesterol chow contains 1% cholesterol and 5% peanut oil. Angll infused to peritoneal cavity by injection syringes (single use) at a dose of 500ng/kg, twice a week on Tuesday and Friday for 16 weeks.2. Animal body weight measurement: The rabbits were weighed at the beginning of the dietary intervention, 8 weeks and 16 weeks after the studyrespectively.3. Serum lipid profile measurement: Respectively at the beginning of the study, 8 weeks and 16 weeks after the study, blood samples were withdrawn from the central ear artery of rabbits. Total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, apolipoprotein Al (ApoA) and apolipoprotein BlOO (ApoB) concentrations were determined and then calculated HDL/LDL ratio, atherosclerotic index AI=(TC-HDL)/HDL and ApoB/ApoA ratio. TC and TG were determined by enzymatic method and HDL, LDL, ApoA and ApoB were determined by immuno-turbidimetry assay respectively.4. Histopathologic evaluation of abdominal aorta4.1 At the end of the study after 16 weeks, the abdomial aorta was dissected and then stained with hematoxylin and eosin (H&E) for light microscopy to observe the vascular atherosclerotic lesion and its degree.4.2 Histomorphometric analysis was processed using the Image-Pro Plus Image Analysis System. The average thickness of the intima (I) and the media (M) of abdominal aorta, the aortic intimal plaque surface (Sp) and the medial surface (Sm) were assessed by using of multiple (n=8 in each group) sections, and then calculated I/M and Sp/Sm-4.3 Immunohistochemical detection of the expression of macrophage and smooth muscular cell (SMC): The expression of macrophage and SMC in the intima of abdominal aorta was detected by SABC (Strept-avidin-biotin Complex) and diaminobenzidine (DAB) staining method. The number ratio of the positive macrophage (macrophage%) and SMC (SMC%) was counted with Image-Pro Plus Image Analysis System respectively.5. Immunohistochemical detection of the expression of LOX-1 protein: The immunostaining for LOX-1 was using ABC (Avidin-Biotin Complex) and DAB staining method. The area ratio of the positive expressing cells of LOX-1 (LOX-1 %) was analyzed with Image-Pro Plus Image Analysis System.6. Western Blot analysis of the expression of LOX-1 protein. DAB staining method.7. Determination of the mRNA expression of LOX-1, MCP-1 and VCAM-1 by RT-PCR method: The relative expressing level was defined by optical density ratio of LOX-1, MCP-1 and VCAM-1 to GAPDH respectively (LOX-1/GAPDH, MCP-1 /GAPDH and VCAM-1/GAPDH).8. Statistical analysis: All data are expressed as mean± standard deviation (SD) and analyzed using statistical package SPSS 11.0 for Windows. One-way analysis of variance (ANOVA) was used to determine statistically significant differences among the three groups and means of every two different groups were compared with Student's t test. Some data were performed with gradually linear correlation analysis. A level of P<0.05 was considered statistically significant. Results:1. Body Weight1.1 In HC-diet indued AS model: At the end of 8 weeks, there was no statistical difference among the four groups but at the end of the study the rabbits of HC-diet group were lighter than rabbits of Control group (P<0.01) and Statin group and Losartan group (P<0.05).1.2 In HC-diet plus Angll injection induced AS model: At the end of 8 weeks, there was no statistical difference among the four groups but at the end of the 16 weeks period the rabbits of HC-control group and HC-injection group were similarly lighter than rabbits of Normal control group (P<0.01) and Normal injection group (P<0.05).2. Serum Lipid Profile2.1 After 8 weeks of dietary intervention, serum TC, LDL, AI and ApoB levels and ApoB/ApoA ratio in HC-diet group were markedly higher while HDL/LDL ratio was significantly lower than that of Control group (P<0.01). At the end of 16 weeks serum lipid continued at high levels, while intervention with fluvastatin and losartan they all decreased significantly (P<0.01 vs HC-dietgroup). The hypolipidemic effect of fluvastatin was more significant than losartan (p<0.05).2.2 After 8 weeks of dietary and AngH intervention, serum TC, LDL, AI and ApoB levels and ApoB/ApoA ratio were markedly higher in HC-control group and HC-injection group compared to Normal control group (P<0.01). At the end of 16 weeks the above parameters kept on high levels in HC-control group, but in HC-injection group, some of them decreased in some degree while AI and ApoB/ApoA ratio increased continuously. There were no statistical changes of lipid profile in rabbits of Normal control group and Normal injection group.3. Aortic Vascular Wall Proliferation3.1 There was no intimal proliferation in Control group. However, in HC-diet group, the intimal proliferation was significant and lipid plaque was widespread, in which foam cells accumulated in mass. Treatment with fluvastatin and losartan similarly attenuated this intimal proliferation markedly, the plaque was limited and the number of foam cells was deduced and its volume was decreased despite administration of high-cholesterol diet.3.2 In light microscopy, the intima was thin and its structure was intact while there was significant proliferation in the media of Normal injection group. In HC-control group and HC-injection group, the intimal proliferation was significant and lipid plaque was widespread, in which foam cells accumulated in mass similarly. These changes were more striking in HC-injection group.4. Histomorphometric Analysis of Aortic Vascular Wall Proliferation4.1 In abdominal aorta of HC-diet group, I thickened, I/M augmented, Sp and Sp/Sm enlarged dramatically (P < 0.01 vs Control group). Treatment with fluvastatin and losartan similarly attenuated the degree of intimal proliferation, reduced atheroslerotic plaque coverage (P<0.01 vs HC-diet group). The effect of fluvastatin was more significant than losartan.The aortic intimal surface involvement (Sp) of HC-diet group rabbits was positively correlated with TC, LDL, AI and ApoB/ApoA ratio while negativelycorrelated with HDL, in which the correlation was most obvious for ApoB/ApoA ratio (P<0.01). However there was no significant correlation between Sp and TG (P>0.05).4.2 Compared to Normal control group, M thickened and Sm enlarged obviously in Normal injection group (P<0.05), while I thickened, I/M augmented, Sp and Sp/Sm enlarged dramatically in HC-control group and HC-injection group (P<0.01). These changes were more dramatical in HC-injection group compared to HC-control group (P<0.05).5. Immunohistochemical analysis of macrophage and a -actin in the intima5.1 Macrophage and SMC-derived foam cells were the main component of atherosclerotic plaque and Macrophage(%) was most dramatic in HC-diet group. Macrophage( %) deduced in different degree in Statin group and Losartan group (P <0.01 vs HC-diet group) while SMC(%) was not different significantly among them.5.2 Macrophage-derived foam cells were the main component cells of atherosclerotic plaque in HC-control group, while the ratio of SMC-derived foam cells increased dramatically in HC-injection group (P<0.01 vs HC-control group).6. The protein expressing level of LOX-1 by immunohistochemistry6.1 There were minimal expression of LOX-1 in the endothelium and neointima of Control group while HC-diet significantly enhanced LOX-1 expression (P<0.01 vs Control group) and its expressing position was mainly in the endothelium and neointima, especially most obvious in the neointima. Treatment with fluvastatin and losartan similarly prevented the enhanced LOX-1 expression dramatically in the aortic wall (P<0.01 vs HC-diet group), but the prevention effect was more striking in Statin group compared to Losartan group(P <0.05).6.2 In HC-diet plus Angll injection induced AS model, LOX-1 expression was augmented most dramatically in Normal control group, which was concentrated in the aortic endothelium. In HC-injection group its expression inaortic endothelium was less than HC-control group (P<0.05), in which increased LOX-1 positive signals were mostly in aortic neointima.7. Western Blot resultsThere was LOX-1 protein expression in all the four groups. Compared with Control group, LOX-1 protein was increasingly expressed in HC-diet group obviously (P<0.01). After treatment with fluvastatin and losartan, its expression level similarly depressed significantly(P<0.01 vs HC-diet group), while the prevention effect was more significant in fluvastatin than losartan (P<0.01).8. LOX-1, MCP-1 and VCAM-1 mRNA Expression by RT-PCR8.1 Based on semi-quantitative RT-PCR, HC-diet increased LOX-1, MCP-1 and VCAM-1 mRNA expression dramatically (P<0.01 vs Control group) and treatment with fluvastatin and losartan strikingly decreased this enhanced LOX-1 mRNA expression (P<0.01 vs HC-diet group). The decrease was more significant in Statin group (P<0.05 vs Losartan group).Correlation analysis showed that in the four groups, LOX-1 mRNA expressing level was positively correlated with TC, LDL, AI, ApoB/ApoA ratio, Sp and the gene expressing level of MCP-1 and VCAM-1.8.2 In HC-diet plus Angll injection induced AS model, LOX-1, MCP-1 and VCAM-1 mRNA expression was augmented most dramatically in HC-injection group (P<0.01 vs Normal control group). Their expression was also increased in HC-control group and HC-injection group, but the augmentation was more obvious in HC-control group (P<0.01 vs HC-injection group).Correlation analysis showed that in the four groups, LOX-1 mRNA expressing level was positively correlated with the gene expressing level of MCP-1 and VCAM-1. Conclusions:1. High-cholesterol diet induced HC and extensive intimal thickening and the atherosclerotic vacluar pathological changes, but it might not increase body weight of rabbits certainly.2. The aortic intimal surface involvement induced by high-cholesterol diet was positively correlated with TC, LDL, AI and ApoB/ApoA ratio and negatively correlated with HDL. This indicated that the formation and development of the atheromatous plaque was correlated closely with dyslipidemia.3. High-cholesterol diet induced the upregulation of LOX-1 expression at gene and protein levels in the aortic vascular wall of rabbits and its mRNA expressing level was positively correlated with TC, LDL, AI, ApoB/ApoA ratio and aortic plaque surface (Sp). This indicated that hyperlipidemia and hyperlipoproteinemia of LDL could increase the expression of the receptor of ox-LDL and so promote or accelerate atherogenesis.4. There was over-expression of MCP-1 and VCAM-1 mRNA in the hypercholesterotic and atherosclerotic lesions induced by high-cholesterol diet and their expressing level was positively correlated with the expressing level of LOX-1 mRNA. This indicated that the expression of LOX-1 was synchronized with the chemoattractive and adhesive process in the atherogenesis and there might have close association between them.5. Single exogenous injection of Angll did not induced typical vascular pathological changes of AS and there was only thickening of aortic media. However, it induced dramatical upregulation of LOX-1 mRNA and protein expressing levels, and at the same time augmented the expressing levels of MCP-1 and VCAM-1 mRNA synchronously. This indicated that Angll was the independent risk factor of AS and it might be involved in the atherogenesis through oxidation and inflammation process.6. The exogenous supply of Angll simutaneously on the basis of high-cholesterol diet accelerated and aggravated the development of diet-induced atherosclerotic pathologyical process. This indicated that Angll and HC were both the independent atherogenic risk factors and at the same time there were synergistic effects between them in the atherogenesis.7. The early intervention of fluvastatin and ATlRBs losartan similarlydiminished serum lipid and retarded the progression of AS effectively in rabbits with diet-induced hyperlipidemia. However, the hypolipidemic and anti-atherosclerotic effect was more significant for fluvastatin than losartan. This indicated that statins should be the first-line choice for the prevention of atherogenesis induced by HC.8. Early prevention with fluvastatin and losartan markedly inhibited the enhanced LOX-1 gene and protein expression in the aortic wall of high cholesterol diet rabbits, and down-regulated MCP-1 and VCAM-1 gene expression synchronously. This indicated that the anti-atherosclerotic actions of statins and ATlRBs were correlated with their antioxidant and anti-inflammation capabilities. Innovations and meanings:1. This study demonstrated in vivo that there was over-expression of LOX-1 mRNA and protein in the aortic wall of rabbits with HC and early AS induced by high cholesterol diet. The correlaion analysis for the first time demonstrated LOX-1 mRNA expressing level was positively correlated with serum lipid (TC, LDL, AI and ApoB/ApoA ratio), aortic intimal surface involvement (Sp) and the gene expressing level of proinflammatory cytokine. It indicated that the expression and its regulation of LOX-1 is an early and key event in the vascular inflammation and atherogenesis induced by HC and it is a major provision to detect and prevent LOX-1 expression earlier for the prevention and therapy of AS.2. This study demonstrated in vivo that single exogenous injection of Angll did not induced typical vascular pathological changes of AS but induced dramatical upregulation of LOX-1 mRNA and protein expressing levels in neointima of aortas of rabbits, and at the same time augmented the expressing levels of MCP-1 and VCAM-1 mRNA synchronously. The exogenous supply of Angll simutaneously on the basis of high-cholesterol diet accelerated and aggravated the development of diet-induced atherosclerotic pathologyical process. It indicated that Angll and HC are both the independent atherogenic risk factorsand at the same time there are synergistic effects between them in the atherogenesis while LOX-1 may be the molecular mechanism for their relation. The prevention on LOX-1 expression could represent a new and potentially significant therapeutic target for interrupting their synergy and for the treatment of AS.3. This study first contrasted and demonstrated that in rabbits with diet-induced AS, fluvastatin and losartan similarly had hypolipidemic and anti-atherosclerotic effects, and also markedly inhibited the enhanced LOX-1, MCP-1 and VCAM-1 expression in the aortic wall. However, the prevention effect was better for fluvastatin compared to losartan. This indicated that statins and ATlRBs have anti-inflammation and antioxidant activities and they are both choices, while statins may be the first-line choice for the early prevention of AS induced by HC.
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