Changes of metallothionein contents in plasma and tissues of rabbit with atherosclerosis

AIM: To observe the level of metallothionein (MT) in liver, aorta and plasma of rabbit with atherosclerosis (AS) in order to recognize the alteration of oxidative defense system in body when AS occurred.METHODS:Preparation of AS model of rabbit induced by having high-fat diet for eight weeks; the levels of MT and malondialdehyde (MDA) were measured in the tissues of liver and aorta and plasma of rabbit.RESULTS:The MT levels in liver tissues and plasma in atherosclerotic group increased 318%(P＜0.01) and 62% (P＜0.01), compared with control group, but its level in aortic tissue in atherosclerotic group decreased 33% (P＜0.01). The MDA levels in liver, aortic and plasma in atherosclerotic group increased 95%(P＜0.01), 76%(P＜0.01) and 42%(P＜0.01), respectively, compared with control group. The changes of contents of MT in liver and plasma have relation with level of MDA in liver tissues and plasma.CONCLUSION:The alteration of MT in liver tissues and plasma in atherosclerotic rabbits may be related to lipid hyperoxidative injury.     

Proliferation and apoptosis of human umbilical vein endothelial cells induced by oxidized low density lipoprotein

AIM:To investigate proliferation and apoptosis of cultured endothelial cell (ECV-304 cell line) induced by varied concentrations of oxidized low density lipoprotein (ox-LDL). METHODS:Cell morphology, Typan blue test, MTT test, LDH release test, flow cytometry and micro-molecular weight DNA fragment gel electrophoresis of apoptosis were used for the detection of the cytotoxic effects of ox-LDL on ECV-304 cell line.RESULTS:0.1, 1, 10 mg/L ox-LDL could promote proliferation of ECV-304 cells. When the concentration of ox-LDL reached up to 100 mg/L and above, the distinct cytotoxic effect appeared. Further study showed that the apoptosis rate of endothelial cells, induced by ox-LDL of 150 mg/L and 200 mg/L for 12 hours, are 15.86% and 21.89%, respectively. 18 h and above hours after incubation, the apoptosis rate began to decrease and rate of necrosis increased. CONCLUSION:ox-LDL has strong cytotoxic effects on endothelial cells and could give rise to different pathologic process, such as proliferation, apoptosis prophase, apoptosis and necrosis.     

Effect of OX-LDL and simvastatin on PKC activity and cytosolic free Ca2+ in cultured rat aortic smooth muscle cells

AMI:To clarify whether OX-LDL and simvastatin can induce the changes of PKC activity and cytosolic free Ca²⁺ in rat aortic smooth muscle cells (ASMC). METHODS:PKC activity and cytosolic free Ca²⁺ were measured by its ability to transfer phosphate from [³²P]ATP to lysine-rich histone and flow cytometric analysis after loading with the Ca²⁺dye fluo-3/Am, respectively. RESULTS:OX-LDL increased PKC total activity in a dose-dependent manner and induced translocation of PKC from the cytosolic to membrane, while OX-LDL induced biphasic [Ca²⁺]i responses including the rapid initial transient phase and the sustained phase. When simvastatin was added, the translocation of PKC was markedly decreased and simvastatin did not impair the initial peak response to OX-LDL but significantly reduced the subsequent plateau phase. CONCLUSSION:OX-LDL can induce dynamic changes of signal transduction of PKC and cytosolic free Ca²⁺ in ASMC and these two events are closely linked.     

Dual roles of oxidized LDL in modulating expression of inflammatory molecules in HUVECs

AIM:To determine the role of LOX-1/PPAR pathway in regulating expression of adhesion molecules elicited by oxidizing low density lipoprotein(Ox-LDL) through Lectin-like oxidized low-density lipoprotein receptor-1(LOX-1) in human umbilical vein endothelial cells(HUVECs). METHODS:HUVECs were incubated with Ox-LDL,poly(I),carrageenan or 15-deoxy-△12,14-prostaglanding J2 (15d-PGJ₂ ). PPAR mRNA and protein were examined by real time RT-PCR and Western blotting. ICAM-1 and E-selectin were detected by RT-PCR and Western blotting respectively. RESULTS:Ox-LDL increased PPAR expression in HUVECs,which was inhibited by pretreatment of HUVECs with LOX-1 blockers. Preincubation of HUVECs with 15d-PGJ₂ attenuated the expression of intercellular adhesion molecule-1(ICAM-1) and E-selectin in response to Ox-LDL. Upregulation of ICAM-1 and E-selectin mediated by Ox-LDL were suppressed more significantly by the combination of 15d-PGJ₂ and polyinosonic acid as compared to either 15d-PGJ₂ or polyinosonic acid alone. CONCLUSION:The results indicate that Ox-LDL exerts a biphasic effects on inflammatory response. It evokes harmful effects by inflammatory injury on one side and protective effects by triggering the LOX-1/ PPAR signaling pathway on the other hand.     

Recent advances in apolipoprotein M and atherosclerosis

Atherosclerosis is a complex pathological process, which has a close relationship with inflammatory response and disorder of lipid metabolism. The cardioprotective role of high-density lipoprotein (HDL) is related to its characters of protecting the vascular endothelial cells and the properties of anti-inflammation and anti-oxidation. Low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) are thought to have the adverse effects on human atherosclerosis. Apolipoprotein M (apoM, found in 1999) is an apolipoprotein mainly associated with HDLs. ApoM has a remarkable property of anti-atherosclerosis in animal experiment. However, recent studies have not yet been able to establish that apoM is a defining risk factor for human coronary heart disease, and the biological functions of apoM, including its potential role in human atherogenesis, need to be established.     

Serum paraoxonase activity in dairy cows during pregnancy

Preparturient dairy cows are at high risk of metabolic and reproductive disorders and oxidative stress is considered to be involved in these events. We investigated the serum paraoxonase activity in dairy cows during pregnancy and alterations in lipid and lipoprotein patterns in this period. The relation between paraoxonase activity and HDL-cholesterol concentration was also compared. The study was carried out on 76 pregnant lactating and 26 pregnant dry Holstein dairy cows. The serum paraoxonase activity was determined by the method of hydrolysing of paraoxon, while triglyceride, cholesterol and HDL-cholesterol concentrations were measured by the enzymatic kit methods. A significantly higher serum triglyceride concentration (P<0.001) was observed in dry cows compared to lactating cows. The total cholesterol and HDL-cholesterol concentrations were significantly lower (P<0.001) in dry cows than in lactating ones. In dry cows, paraoxonase activity was significantly lower than in those lactating (P<0.001). There was no significant difference in paraoxonase/HDL-cholesterol ratio between the investigated groups. It seems that the lower HDL concentration could be one of the causes of reduced paraoxonase activity considering the role of HDL as a carrier of most paraoxonase molecules in the blood. A decreased serum paraoxonase activity could diminish the effectiveness and total capacity of the whole antioxidative system during prepartum period in dairy cattle.     

Association of endothelial lipase gene Thr111Ile and Gly26Ser polymorphism with lipoprotein in patients with coronary heart disease

AIM: To investigate the association of endothelial lipase gene (LIPG) Thr111Ile and Gly26Ser polymorphism with lipoprotein in patients with coronary heart disease (CHD) in Chinese.METHODS: 438 patients were classified as 242 CHD group and 196 controls group by selective coronary angiography.Plasma level of lipoprotein was determined and the Thr111Ile and Gly26Ser polymorphism was screened by PCR-RELP.RESULTS: The frequencies of Thr111Ile genotype in Chinese were CC 76.7%,CT 23.3％,TT 0.0％.The frequencies of allele were C 88.3％,T 11.7%.The plasma level of HDL-c in CT group was significantly higher than that in CC group (P<0.05) on logistic regression analysis.However,logistic regression analysis revealed that there was no significant difference between CHD group and control group for Thr111Ile polymorphism (P>0.05).No Gly26Ser mutation was observed in this study.CONCLUSION: The polymorphism of Thr111Ile is present in patients with CHD in Chinese,and T allele is related to high HDL-c level.There is no significant association between the polymorphism of Thr111Ile and CHD.The Gly26Ser mutation has not found in this study.     

Effects of phospholipid transfer protein overexpression on content of sphingosine-1-phosphate in mouse lipoprotein

AIM：To investigate the interaction and the mechanism of sphingosine-1-phosphate (S1P) and phospholipid transfer protein (PLTP) in lipoprotein. METHODS：The S1P content in the plasma and lipoprotein from 10-week-old PLTP transgenic (PLTP-Tg) mice and wild-type (WT) mice (n=8 each) was assayed. The transport of S1P by PLTP was determined by S1P transfer assay. The content of specific S1P carrier, apolipoprotein M, was detected by Western blotting. RESULTS：Plasma S1P contents were decreased by 21.1% in PLTP-Tg mice compared with WT mice. S1P content in high-density lipoprotein (HDL) fraction (HDL-S1P) from PLTP-Tg mice was decreased by 35.1% compared with WT mice, whereas the S1P in low-density lipoprotein (LDL) fraction (LDL-S1P) was increased by 127.4%. The results of S1P transfer assay indicated that PLTP facilitated S1P transport from erythrocyte to recombinant liposome at 37 ℃ in D-Hanks buffer solution. The plasma content of apolipoprotein M was not changed in PLTP-Tg mice compared with WT mice. CONCLUSION：PLTP is a key factor to maintain plasma HDL-S1P under physical condition. Overexpression of PLTP decreases the HDL-S1P but increases LDL-S1P. The mechanism might be related to the capability of PLTP on transferring S1P from erythrocyte to lipoprotein.     

Comparative study of lipoprotein monosaccharide composition between diabetic patients and healthy participants

AIM:To establish a liquid chromatography method for determining the monosaccharide composition of human lipoproteins, and to investigate the differences between diabetic patients and healthy participants. METHODS:Liquid chromatography with pre-column derivatization was used to determine the neutral and basic monosaccharides, and liquid chromatography tandem mass spectrometry was applied to quantify N-acetylneuraminic acid content. RESULTS:The contents of mannose, glucosamine, N-acetylglucosamine, glucose, galactose and N-acetylneuraminic acid in high-density lipoprotein from healthy participants and diabetic patients were (5.88±0.94),(16.49±4.11),(1.31±0.33), (0.87±0.16), (7.18±1.64), (2.14±0.12) mmol/(g protein) and （8.68±0.39), (24.73±5.50), (1.91±0.54), (1.23±0.35), (9.73±2.85), (3.53±0.27） mmol/(g protein）, respectively. The contents of mannose, glucosamine,N-acetylglucosamine, glucose, galactose and N-acetylneuraminic acid in low-density lipoprotein from healthy participants and diabetic patients were (29.20±3.57), (50.77±4.72), (5.28±0.64), (10.06±1.37), （28.44±3.96）,（6.86±0.11) mmol/(g protein) and (30.08±3.78）, （38.52±6.38）, （3.79±0.78）, （7.63±1.50）, （20.05±2.63）, （6.45±0.18) mmol/(g protein), respectively. The contents of mannose, glucosamine, glucose, galactose and N-acetylneuraminic acid in very-low-density lipoprotein from healthy participants and diabetic patients were (91.21±4.12）, （27.05±2.34）, （4 230.95±15.83）, (43.40±3.75), (2.95±0.24) mmol/(g protein) and (82.40±0.51), (30.16±0.32), (4 722.73±93.27), (34.05±2.81), (4.42±0.15) mmol/（g protein）, respectively. CONCLUSION: Liquid chromatography with pre-column derivatization is suitable for the neutral and basic monosaccharide analysis in human lipoproteins, and the glycosylation of lipoproteins in diabetic patients are significantly changed compared with the healthy controls.