Role Of Lectin-like Oxidized Low-density Lipoprotein Receptor-1 In The Progress Of Atherosclerosis Enhanced By Advanced Glycation End Products

PART I Effects of AGEs on Expression of LOX-1 in Human Vascular Endothelial Cells and its MechanismObjective:To investigate the effect of advanced glycation end products (AGEs) on expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in cultured human umbilical vein endothelial cells (HUVECs), and the influence of rosiglitazone and antisense LOX-1 mRNA on LOX-1 expression. To explore the role of LOX-1 in the progress of atherosclerosis enhanced by AGEs, and its possible mechanism.Methods:AGEs were made by incubating bovine serum albumin(BSA) with high concentration of glucose in 37℃ in vitro. HUVECs were cultured according to the descriptions in literature. The activities of SOD and the level of MDA were detected by spectrophotometry. HUVECs were treated with AGE-BSA (0,50,100,200,400 mg/1) for 24 h or AGE-BSA (200 mg/1) for 0,8,16,24,48,72h respectively. HUVECs were exposed to AGE-BSA of 200 mg/1 with different concentrations of rosiglitazone and antisense LOX-1 mRNA for 24 hours. The mRNA and protein of LOX-1 and ICAM-1 were analyzed by RT-PCR and westernblot respectively. Adhesion assay of peripheral blood mononuclear cells (PBMCs) to HUVECs was performed, and adhesion rate of PBMCs to HUVECs was also measured.Results:AGE-BSA inhibited the activities of SOD and increased MDA level in supernatants (P<0.05). AGE-BSA increased the expression of LOX-1 in the level of mRNA by the time-and dose-related manner (P<0.05). Rosiglitazone and antisense LOX-1 mRNA decreasd the expression of mRNA and protein in HUVECs treated with AGE-BSA by the manner of concentration (P<0.05). Rosiglitazone and antisense LOX-1 mRNA also decreasd the expression of ICAM-1 mRNA and protein in HUVECs treated with AGE-BSA and markedly depressed the adhesion of HUVECs to PBMCs (P<0.01).Conclusions:AGEs could induce more production of reactive oxygen species and inhibit the activities of SOD, which results in accelerating the progress of atherosclerosis. AGEs can upregulate the expression of LOX-1 mRNA and protein in HUVECs, the upregulation were suppressed by rosiglitazone and antisense LOX-1 mRNA, Rosiglitazone and antisense LOX-1 mRNA also decreasd the expression of ICAM-1 mRNA and protein in HUVECs treated with AGE-BSA and markedly depressed the adhesion of HUVECs to PBMCs. These findings suggest that LOX-1 might play an important role in the progress of atherosclerosis enhanced by AGEs. And the possibly mechanisms were oxidative imbalance and increasd expression of ICAM-1. Rosiglitazone has the role of anti-atherosclerosis besides its hypoglycemic effect.Part Ⅱ Effects of AGEs on Expression of LOX-1 on Aorta of Rabbit with High Fat Diet and the Intervention Effects of DrugsObjective:To investigate the effects of advanced glycation end products(AGEs) on expression of LOX-1 on aorta of rabbit with high fat diet, and the intervention effects of rosiglitazone and rosuvastatin. To explore the pathway of atherosclerosis promoted by AGEs, and to explore the anti-atherosclerosis role of rosiglitazone and rosuvastatin.Methods: Forty two New Zealand white rabbits with high fat diet were divided randomly into 6 groups:group A, feed with normal diet (n=7); group B, feed withs high fat diet(n=7); group C, fed with high fat and injected intravenously with AGE modified rabbit serum albumin (AGE-RSA,5 mg/kg, once every other day) (n=7); group D, fed with high fat and injected intravenously with unmodified RSA (RSA,5 mg/kg, once every other day)(n=7); group E, fed with high fat and injected intravenously with AGE modified rabbit serum albumin model (AGE-RSA,5mg/kg, once every other day), fed rosiglitazone 0.5mg/(kg-d) additional (n=7); group F, fed with high fat and injected intravenously with AGE modified rabbit serum albumin model (AGE-RSA,5mg/kg, once every other day), fed rosuvastatin 1.5mg/(kg-d) additional (n=7). Ten weeks after the rabbits were sacrificed, their abdominal aortas were taken out and stained with oil red O; HE staining were used to detect the morphology and structural changes of vascular under light microscope; immunohistochemical staining were used to test the content of LOX-1 in aortic tissue; blood lipids and hs-CRP were checked after the rabbits were sacrificed. Westernblot were used to detect the expression and content of NF-κB p65 protein in the nucleus of vascular tissue.Results:1. The results of Oil red O staining and HE staining shows that compared with group A, the plaque area and intima-media thickness in group B were significantly increased (P<0.01). Compared with group B and group D, the plaque area and intima-media thickness in group C were increased (P<0.05). Compared with group C, after the treatment with rosiglitazone and rosuvastatin, both the plaque area and intima-media thickness in group E and group F were decreased (P<0.05). There were no significantly deference between group E and F (P>0.05).2. The results of immunohistochemical staining show that in group A, there were no significant LOX-1 positive cells in aortic tissue; compared with group B and D, the LOX-1 positive cells increased significantly in aortic tissue of group C (P<0.01). Compared with group C, after the treatment with rosiglitazone and rosuvastatin, the LOX-1 positive cells decreased significantly in aortic tissue (P<0.05), there were no significant difference between group E and group F (P>0.05).3. The results of serum detection show that compared with group A, the levels of CHO and LDL-C in group B, group C and group D were increased (P<0.01), but there were no significantly deference between group D and group C (P>0.05). Compared with group C, after the treatment with rosuvastatin, the levels of CHO and LDL-C in group F were decreased(P<0.05); While after the treatment with rosiglitazone, no significantly changes were found for the levels of CHO and LDL-C in group F. Compared with group A, the levels of TRG in group B, group C and group D had a upward trend, but there were no significant statistical difference (P>0.05). Compared with group C, after the treatment with rosuvastatin, levels of TRG in group F had a downward trend, but there were no significant statistical difference (P>0.05). Compared with group B and D, the levels of hs-CRP in group C were significantly increased (P<0.01). Compared with group C, after the treatment with rosiglitazone and rosuvastatin, levels of hs-CRP in group E and F were significantly decreased (P<0.01), but no significantly deference were found between group E and group F (P>0.05).4. The results of westernblot show that compared with the group B and group D, the levels of NF-κB p65 protein in the nucleus of vascular tissue in group C significantly increased (P<0.01).Compared with group C, after the treatment with rosiglitazone and rosuvastatin, NF-κB p65 protein in group E and group F decreased (P<0.05). However, there were no significant difference between group E and group F (P>0.05).ConclusionsAGEs can increase aortic plaque area and intima-media thickness for rabbits with high fat diet; AGEs accelerated the formation of atheromatous plaque, and promoted the expression of LOX-1 on aorta, which indicated that LOX-1 may play an important role in the progress of atherosclerosis. Rosiglitazone and rosuvastatin can attenuate atheromatous plaque area and intima-media thickness, decrease NF-κB p65 protein in the nucleus of vascular tissue, reduce the expression of LOX-1 on aorta and lower the levels of hs-CRP in serum, which shows that rosiglitazone and rosuvastatin play the roles of anti-atherosclerosis by depressing of vascular inflammation, reducing the expression of NF-κB p65 in the nucleus of vascular tissue and LOX-1 on aorta.Part Ⅲ Values of sLOX-1 and hs-CRP in patients with acute coronary syndrome and the Intervention Effect of DrugsChapter one Values of soluble lectin-like oxidized low density lipoprotein receptor-1 and high-sensitivity C-reactive protein in the patients with acute coronary syndromeObjectiveTo investigate the serum levels of soluble lectin-like oxidized low-density lipoprotein receptor-1(sLOX-1)and high-sensitivity c-reactive protein (hs-CRP) in patients with coronary artery disease for different types and their correlation, and to explore the prediction function of sLOX-1 and hs-CRP in patients with acute coronary syndrome(ACS).Methods:Enzyme-linked immunosorbent assay was used to measure the sLOX-1 and hs-CRP levels in 62 patients with coronary artery disease(34 with ACS and 28 with stable angina pectoris)and 27 controls.Results:In the patients with ACS serum sLOX-1 and hs-CRP levels were significantly higher than those in control group (P<0.01) and stable angina pectoris group (P<0.05). In patients with stable angina pectoris serum sLOX-1 and hs-CRP levels were significantly higher than those in control group (P<0.05). Significantly positive correlation between the serum levels of LOX-1 and hs-CRP was observed in the patients with acute coronary syndrome (r=0.927, P<0.001).Conclusions:Serum sLOX-1 and hs-CRP levels were elevated in ACS patients, which shows the possible relation to the pathogenesis of ACS and may serve as a potential marker of plaque instability.Chapter two Effects of loading-dose rosuvastatin on serum sLOX-1, hs-CRP, LVEF and recent prognosis in patients with acute coronary syndromes undergoing elective percutaneous coronary interventionObjectiveTo investigate the effects of loading-dose rosuvastatin on serum levels of lectin-like oxidized low-density lipoprotein receptor-1(LOX-1), high-sensitivity c-reactive protein (hs-CRP), LVEF and recent prognosis in patients with non-ST segment elevation acute coronary syndromes (NSTEACS) undergoing elective percutaneous coronary intervention(PCI).Methods:A total of 72 patients with non-ST segment elevation ACS were randomized to the loading-dose group pretreated with 20 mg rosuvastatin 12 hours before PCI and with a second dose administered 2 hours before PCI (n=33) and control group treated with standard method according guideline (n=39). Serum levels of sLOX-1, hs-CRP, CK-MB and cTnI were measured prior to PCI, and at 24 hours and 30 days after PCI.The levels of LVEF were also recorded prior to PCI and 30days after PCI in both groups.30-day incidence of major adverse cardiac events was recorded in two groups.Results:Compared to pre-PCI, serum levels of sLOX-1 and hs-CRP of the two groups were increased at 24hours after PCI (all P<0.05), the levels of CK-MB and cTnI were also increased (P<0.01), however, the increased values of sLOX-1, hs-CRP, CK-MB and cTnl were significantly lower in loading-dose rosuvastatin-treated group than in control-treated group; Serum levels of sLOX-1 and hs-CRP were lower in loading-dose rosuvastatin-treated group than in control-treated group at 30days after PCI (P<0.05).Serum levels of TC and LDL-C were not changed at 24hours after PCI (P>0.05) until 30days after PCI (P<0.05), but there were no difference between the two groups. No significant difference was found for serum levels of ALT and Scr between the two groups before and after PCI. Compared to the control-treated group, serum levels of BNP decreased (P<0.05) and LVEF (P<0.05) increased in the loading-dose rosuvastatin-treated group. The 30-day incidence of MACE occurred in 6.06% of patients in loading-dose group and 23.08% of patients in control group (P<0.05).ConclusionsThe therapy of loading-dose rosuvastatin for patients with non-ST segment elevation acute coronary syndromes (NSTEACS) undergoing elective percutaneous coronary intervention can attenuate the increase of serum levels of sLOX-1 and reduce myocardial injury and inflammatory reaction caused by PCI, and also reduce the occurrence of MACE in 30days after PCI.