Influence of Oxidized Low Density Lipoprotein on the Proliferation of Human Artery Smooth Muscle Cells in vitro

The effects of oxidized low density lipoprotein （ox-LDL） on the proliferation of cultured human vascular smooth muscle cells （vSMC） were investigated in vitro. By using NaBr density gradient centrifugation, LDL was isolated and purified from human plasma. Ox-LDL was produced from LDL by being incubated with CuSO4. ox-LDL was then added to the culture medium at different concentrations （35, 60, 85, 110, 135 and 160μg/mL） for 7 days. The influence of ox-LDL on vSMC proliferation was observed in growth curve, mitosis index, and in situ determination of apoptosis. The data were analyzed with SPSS 10.0 software. The results showed that the ox-LDL produced in vitro had a good purity and optimal oxidative degree, which was similar to the intrinsic ox-LDL in atherosclerotic plaque, ox-LDL at a concentration of 35 μg/mL demonstrated the strongest proliferation inducement, and at a concentration of 135 μg/mL, ox-LDL could inhibit the growth of vSMC. ox-LDL at concentrations of 35 and 50 μg/mL presented powerful mitotic trigger, and with the increase of ox-LDL concentration, the mitotic index of vSMC was decreased gradually, ox-LDL at higher concentrations promoted more apoptotic vSMCs, ox-LDL at lower concentrations triggered proliferation of vSMCs, and at higher concentrations induced apoptosis in vSMCs, ox-LDL played a promotional role in the pathogenesis and development of atherosclerosis by affecting vSMC proliferation and apoptosis.

Effects of Oxidized Low Density Lipoprotein on Transformation of Valvular Myofibroblasts to Osteoblast-like Phenotype

In order to investigate the roles of Wnt signal pathway in transformation of cardiac valvular myofibroblasts to the osteoblast-like phenotype, the primary cultured porcine aortic valve myofibroblasts were incubated with oxidized low density lipoprotein（ox-LDL, 50 mg/L）, and divided into four groups according to the ox-LDL treatment time： control group, ox-LDL 24-h group, ox-LDL 48-h group, and ox-LDL 72-h group. Wnt signal pathway blocker Dickkopf-1（DDK-1, 100 μg/L） was added in ox-LDL 72-h group. The expression of α-smooth muscle actin（α-SMA）, bone morphogenetic protein 2（BMP2）, alkaline phosphatase（ALP）, and osteogenic transcription factor Cbfa-1 was detected by Western blotting, and that of β-catenin, a key mediator of Wnt signal pathway by immunocytochemical staining method. The Wnt/β-catenin was observed and the transformation of myofibroblasts to the osteoblast-like phenotype was examined. The expression of α-SMA, BMP2, ALP and Cbfa-1 proteins in the control group was weaker than in the ox-LDL-treated groups. In ox-LDL-treated groups, the protein expression of α-SMA, BMP2, ALP, and Cbfa-1 was significantly increased in a time-dependent manner as compared with the control group, and there was significant difference among the three ox-LDL-treated groups（P〈0.05 for all）; β-catenin protein was also up-regulated in the ox-LDL-treated groups in a time-dependent manner as compared with the control group（P〈0.05）, and its transfer from cytoplasm to nucleus and accumulation in the nucleus were increased in the same fashion（P〈0.05）. After addition of DKK-1, the expression of α-SMA, bone-related proteins and β-catenin protein was significantly reduced as compared with ox-LDL 72-h group（P〈0.05）. The Wnt/ β-catenin signaling pathway may play an important role in transformation of valvular myofibroblasts to the osteoblast-like phenotype.

Obstructive jaundice leads to accumulation of oxidized low density lipoprotein in human liver tissue

Oxidized low density lipoprotein （ox-LDL） molecule is one of the most important modified lipoproteins produced during the oxidative stress. Modified lipoproteins have been defined as being part of the immune inflammatory mechanisms in association with oxidant stress. We have reported the accumulation of ox-LDL in Balb/c mice liver after bile duct ligation previously. Here, we investigated this finding in human beings with obstructive jaundice. Our study demonstrates that obstructive jaundice results in tremendous accumulation of ox-LDL in the liver tissue of patients.

Oxidized low density lipoprotein (Ox-LDL) impacts on erythrocyte viscoelasticity and its molecular mechanism

Aim:The oxidized low-density lipoprotein(OxLDL) plays an important role in atherosclerosis yet it remains unclear if it damages circulating erythrocytes. Method: In this study。

Effects of oxidized low density lipoprotein on the growth of human artery smooth muscle cells

Background Studies have shown that oxidized low density lipoprotein （ox-LDL） promotes the pathogenesis and development of atherosclerosis （AS）, and that the proliferation, migration and phenotype alteration of vascular smooth muscle cells （vSMCs） into foam cells are critical changes in AS. It is proposed that ox-LDL might play a novel role in the pathologic process of vSMCs. The present study was performed ex vivo to investigate the effects of ox-LDL on the growth of cultured human vSMCs.Methods Using NaBr density gradient centrifugation, LDL from human plasma was isolated and purified. ox-LDL was produced from LDL after being incubated with CuSO4. ox-LDL was then added to the culture medium at different concentrations （25μg/ml, 50μg/ml, 75 μg/ml, 100μg/ml, 125μg/ml, and 150μg/ ml） for 7 days. The influence of ox-LDL on vSMC growth was observed from several aspects as growth curve, mitosis index, lipid staining, and in situ determination of apoptosis. The digital results were analyzed with SPSS 10.0.Results The ox-LDL produced ex vivo had a good purity and optimal oxidative degree, which was similar to the intrinsic ox-LDL in atherosclerotic plaque, ox-LDL at a concentration of 25 μg,/ml demonstrated the strongest proliferation. At the concentration of 125 μg,/ml, ox-LDL suppressed the growth of vSMCs. At concentrations of 25 μg/ml and 50 μg/ml, ox-LDL presented powerful mitotic trigger. When the concentration of ox-LDL increased, the mitotic index of vSMCs decreased gradually, ox-LDL induced more foam cells from vSMCs with rich intracellular lipid accumulation at concentrations of 25μg/ml and 50μg/ml, ox-LDL at higher concentrations induced more apoptotic vSMCs.Conclusions ox-LDL at lower concentrations may trigger proliferation and phenotype alteration into foam cells of vSMCs, and at higher concentrations it may induce apoptosis in vSMCs, ox-LDL plays an important role in the pathogenesis and development of atherosclerosis by its effect on vSMCs proliferation, phenotype alteration and apoptosis.

Association between circulating oxidized low-density lipoprotein and atherosclerotic cardiovascular disease

Atherosclerosis is a chronic, progressive disease which eventually leads to coronary heart disease (CHD), ischemic stroke and other atherosclerotic cardiovascular disease (ASCVD). Numerous studies have demonstrated an atherogenic role of oxidized low-density lipoprotein (ox-LDL) in the progression of ASCVD. This article briefly reviews the atherogenic mechanism of ox-LDL, the methods of measuring ox-LDL in the circulation, effect of medical therapy and life-style modification on ox-LDL level, and the association between circulating ox-LDL and atherosclerosis, including clinical ASCVD events and subclinical atherosclerosis, in observational studies.

BLOOD PLATELETS AND OXIDIZED LOW DENSITY LIPOPROTEIN

Functional morphological alterations of human blood platelets induced by oxidatively modified low density lipoprotein (LDL) were studied with an in vitro model by means of electron microscopy, reflection contrast microscopy and quantitative image analysis. The oxidized LDL (50-300 μg/ml) induced the disc-sphere transformation of platelets, the formation of pseudopodia, centralization of granules and degranulation. Platelet plasma membrane was damaged by oxidized LDL leading to a lower electron density of he cytoplasm compared to controls. In an incubation chamber, oxidized LDL-treated platelets sedimented onto the

Erythrocyte membrane Ca^(2+)-ATPase inhibited by oxidized low density lipoprotein

Oxidized low density lipoprotein (LDL) has been shown to inhibit the activity of platelet plasma mambrane Ca^2+ ATPase, and induce changes of factin in endothelial cells. Since the deformability of human erythrocytes are closely related to the calcium metabolism and the stability of cytoskeleton, the influence of oxidized LDL on plasma membrane Ca^2+ ATPase and plasma membrane fluidity were studied in human erythrocytes. Our in vitro experiments demonstrated that oxidized LDL, but not native LDL, inhibits the activity of Platelet Research Unit, Institute of Anatomy, University of Münster, Germany (Zhao B, Filler TJ and Dierichs R) Shanghai Fourth People's Hospital, Shanghai, China (Yu H) Department of Biochemistry, University of Louisville, Kentucky, USA (Dean W)Ca^2+ ATPase in purified erythrocyte plasma membrane (P<0.01). Since no change in the membrane fluidity was detected in the erythrocyte plasma membrane exposed to native LDL and oxidized LDL as estimated by steady state by trimthylammonium diphenylhexatriene (TMA DPH) anisotropy, oxidized LDL did not affect the Ca^2+ ATPase by grossly changing the erythrocyte deformability probably by interfering the cytosolic calcium and then altering the cytoskeleton. The deformability of erythrocytes is important in microcirculation. A better understanding of the interactions between oxidized LDL, erythrocytes, platelets and endothelial cells can prevent cardiovascular diseases.

EFFECT OF OXIDIZED-LDL ON NF-κB NUCLEAR TRANSLOCATION IN AORTIC SMOOTH MUSCLE CELLS ORIGINATED FROM RATS OF DIFFERENT AGES

Objective To investigate the molecular mechanism of atherosclerosis that related to age. Methods Immunohistochemistry staining and Western blot were adopted to determine the nuclear translocation of nuclear factor-kappa B (NF-κB) and expression of platelet-derived growth factor B (PDGF-B) in smooth muscle cells (SMCs) co-cultured with low density lipoprotein (LDL), oxidized LDL (ox-LDL), and ox-LDL+high density lipoprotein (HDL) originated from rats of 2 and 10 months old respectively. Fat stain was used to identify the lipid intake in SMCs. Results The optimal stimulation time of ox-LDL to SMCs was 12 hours. NF-κB intensity increased in most nuclei of SMCs that originated from rats of either 2 or 10 months old co-cultured with ox-LDL. The intensity of NF-κB and the amount of intracellular lipid taken in SMCs were more obvious in cells from 10-month-old rats than from the younger ones. Change of PDGF-B expression in SMCs was not remarkable in each group of rats. Conclusions The 10-month-old rats are more susceptive to ox-LDL than 2-month-old rats in activating nuclear transloca- tion of NF-κB. Maybe this is one of the important reasons contributing to the difference between the older and younger rats on the initiation and development of atherosclerosis lesion. Expression of PDGF-B is not associated with the activity of nuclear translocation of NF-κB.

Mitofusin2 Decreases Intracellular Cholesterol of Oxidized LDL-Induced Foam Cells from Rat Vascular Smooth Muscle Cells

Mitofusin2 （Mfn2） plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells （VSMCs）. The purpose of this study was to investigate the effects of Mfn2 on the traffick- ing of intracellular cholesterol in the foam ceils derived from rat VSMCs （rVSMCs） and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 （ABCA1）, adenosine triphosphate-binding cassette subfamily G member 1 （ABCG1） and peroxisome proliferator-activated receptor gamma （PPARy）. The rVSMCs were co-cultured with oxi- dized low density lipoprotein （LDL, 80 ~tg/mL） to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers （20, 40 and 60 pfu/cell） of recombinant adenovirus containing Mfn2 gene （Adv-Mfn2） were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group （P〈0.05）, and the ex- pression levels significantly increased when the titer of Adv-Mfn2 increased （P〈0.05）. At 24 or 48 h af- ter oxidized LDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased （P〈0.05）, but it was sig- nificantly decreased in Adv-Mfn2-transfected group as compared with untransfected group （P〈0.05）, and it also significantly decreased when the titer of Adv-Mfn2 increased （P〈0.05）. The mRNA and pro- tein expression levels of ABCA1 and ABCG1 were significantly increased in Adv-Mfn2-transfected group as compared with untransfected group （P〈0.05）. Though the mRNA and protein expression levels of PPARy was not significantly increased （P〉0.05）, the phosporylation levels of PPARy were signifi- cantly decreased in Adv-Mfn2-transfected group as compared with untransfected group （P〈0.05）. These results suggest that the transfection of Adv-Mfn2 can significantly reduce intracellular cholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPAR＇/phosporylation and then increasing pro- tein expression levels of ABCAI and ABCG1, which may be helpful to suppress the formation of foam cells.

Effects of IGF-1 and oxLDL on expression of phosphatase PHLPP1 in vascular smooth muscular cells

Objective To investigate the effects of insulin-like growth factor-1 （IGF-1） and oxidized low density lipoprotein （oxLDL） on expression ofphosphatase PHLPP 1 in vascular smooth muscle cells （VSMCs）. Methods Rabbit aortic VSMCs were cultured. VSMCs proliferation ability was determined by measuring cell number and mitochondrial dehydrogenase （MD） activity with MTT assay. Western blot was used to detect the protein expression ofphosphatase PHLPP1. Results IGF-1 （100ug/L） increased cell number and MD activity to 3.02 and 3.59 times of that in control group, oxLDL（501xg/ml） elevated the above two parameters to 2.03 and 2.91 times respectively. Western blot showed that IGF-1 and oxLDL inhibited the expression of PHLPPI to 39.27% and 40.26% of the control group （P〈0.01 ）. Conclusion IGF- 1 and oxLDL may enhance the proliferation of VSMCs by decreasing the expression ofphosphatase PHLPP 1.

Activation of Proteinkinase ERK Mediates Induction of Macrophage MMP-12 by OxLDL

The present study was undertaken to investigate the effect of oxidized low density lipoprotein (oxLDL) on the expression of macrophage matrix metalloproteinase-12 (MMP-12), and the possible mechanisms. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was detected by Western blot Analysis. Enzymatic activity of MMP-12 was determined by β-casein zymography. RT-PCR analysis was used to measure the mRNA expression level of MMP-12. OxLDL-stimulated macrophages produced increased casein-degrading activities and oxLDL also significantly increased the mRNA level of MMP-12 in a dose-dependent manner. OxLDL stimulated the phosphorylation of ERK1/2 in macrophages. The use of the specific inhibitor indicated that the ERK1/2 signaling pathway was required for the induction of MMP-12. These data demonstrated that oxLDL induced MMP-12 expression in macrophages through an ERK1/2-dependent pathway. Key words oxidized low density lipoproteins - macrophage - matrix metalloproteinases CLC number Q 556+.9 Foundation item: Supported by the Natural Science Foundation of Hubei Province (99J138)Biography: He Chun-yan (1970-), female, Ph. D candidate, research direction: biochemistry diagnosis of atherosclerosis.

OxLDL－induced Cytotoxicity and Its Inhibition by API_（0134） in Cultured Porcine Aortic Endothelial Cells

Protective effects of API0134 on endotheliai cells (EC) damaged by oxidatively modified low density lipoprotein (oxLDL) were studied. The results showed that the content of endothelia (ET) and malondialdehyde (MDA) in the media of porcine aortic EC incubated with oxLDL were increased and the cGMP was decreased significantly, and the activity of superoxide dismutase (SOD) was inhibited. The effect of cytotoxicity of oxLDL can be eliminated by API0134.These results suggest that API0134 may protect EC against damages elicited by oxLDL.

NF-κB involved in transcription enhancement of TGF-β1 induced by Ox-LDL in rat mesangial cells

Background To determine the binding activity of nuclear factor-KB (NF-KB) and the transcription of transforming growth factor-pi (TGF-β1) induced by oxidized low density lipoprotein (Ox-LDL) in rat mesangial cells and to elucidate the mechanism of renal injury of Ox-LDL.Methods NF-KB binding activity was measured by gel shift assay in mesangial cells with or without inducement of Ox-LDL. Protein kinase inhibitors and activtors were then used to determine the signal transduction pathways. In this course IKB protein expression was analyzed by Westerm blot assay. TGF-β1 mRNA was measured in mesangial cells exposed to Ox-LDL by RT-PCR assay. TGF-β1 promoter from -1551 to +57 were constructed into a pGL3-Basic vector with a luciferase reporting gene. A putative binding site of NF-KB was mutated. The wild and mutant promoters activity was analyzed by transfection into mesangial cells.Results NF-KB was activated by Ox-LDL persistently and rebounded in the early period. Ox-LDL induced NF-KB activation in a dose dependent way. It also induced IKB degradation in 2 hours and resumed to normal levels. NF-KB activation was not alleviated by inhibitors of protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and p38 MAP kinase (p38MAPK). Inhibitors of protein kinase C (PKC) and proteinsome inhibited the enhancement of NF-KB binding activity. Ox-LDL induced the transcription of TGF-β1 in a time and dose dependent manner. Mutation of the putative binding site of NF-KB reduced the activity of TGF-β1 promoter.Conclusion Ox-LDL induced activation of NF-KB persistently. It was probably regulated by the degradation of IkB mediated by PKC pathway. NF-KB may be involved in the enhancement of TGF-β1 induced by Ox-LDL in rat mesangial cells.

Effects of Suxiao Jiuxin Pill (速效救心丸) on Oxidative Stress and Inflammatory Response in Rats with Experimental Atherosclerosis

Objective: To observe the preventive role of Suxiao Jiuxin Pill (SX速效救心丸) on atherosclerosis (AS) and to probe into the mechanism in the atherosclerosis rat model. Methods: The AS rat model was established by a high fat diet and a large dose of calcium (vitamin D3, 0.6 million U/kg, i.p, once). Sixty healthy male adult Sprague-Dawlay (SD) rats were randomly divided into 6 groups, a normal control group (N), a model group (M), a SX low dose group (SXL), a SX middle dose group (SXM), a SX high dose group (SXH), and an atorvastatin group (ATO) (n=10 in each group). The rats in the treatment groups were given with the specific drugs from the first day by oral administration, and the normal control group and the model group were given with normal saline for 12 weeks. Afterwards, the content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and the content of oxidized low density lipoprotein (ox-LDL) in the serum were detected. In addition, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) proteins were tested by Western-blot method. Results: The serum ox-LDL and MDA level significantly decreased, SOD activity increased in the SX middle, high dose groups and the atorvastatin group compared to the model group (all P<0.05). While the expression of PPARγ and NF-κb proteins significantly decreased in the SX low, middle, high dose groups and the atorvastatin group compared to the model group (all P<0.01), with the best effect in the SX high dose group .These results indicate that SX could elevate the activity of serum SOD, decrease serum level of MDA and ox-LDL, and reduce the expression of PPARγ and NF-κB proteins. Conclusion: SX plays an important role in anti-inflammation and inhibition of oxidative stress, which possibly are the mechanism of its preventing and treating atherosclerosis.

Elevated peroxidative glutathione redox status in atherosclerotic patients with increased thickness of carotid intima media

Background Atherosclerosis is a chronic inflammatory disease. Accumulated evidences suggest a deep involvement of oxidative damage in the development of atherosclerosis, but little is discussed over the relationship between plasma glutathione redox status as the most important intrinsic antioxidant defensive mechanism and the atherosclerosis. Methods A total of 132 patients suspected with atherosclerosis were assigned to three groups by high frequency ultrasonic examination of the carotid artery. With the thickness of intima of the carotid artery as an index of degree of atherosclerosis progression, 56 were included in plaque-forming group （A）, 42 in carotid artery intima-thickening group （B）, and 34 in normal carotid artery intima-thickness group （C）. All patients were subjected to the measurement of plasma glutathione （GSH） （reduced form GSH and oxidized form GSSG）, nicotinamide adenine dinucleotide phosphate （NADP） （reduced form NADPH and oxidized form NADP^＋）, oxidized low density lipoprotein （oxLDL）, and malondialdehyde （MDA） The GSH/GSSG and NADPH/NADP^＋ redox potentials were calculated according to the Nernst equation, and their correlation with intima thickness and oxLDL was analyzed. Results With the thickening of artery intima （from group C to A）, GSH concentration and the ratio of GSH/GSSG gradually reduced, and GSSG and GSH/GSSG redox potential gradually increased （more positive） （P 〈0.05）. The NADPH and NADPH/NADP^＋ redox status also showed similar but milder changes. The products of oxidative stress oxLDL and MDA increased significantly along with the thickening of artery intima （P 〈0.05）. The analysis of the relationship between GSH/GSSG redox potential, intima thickness, and oxLDL showed positive correlations （P 〈0.05）. The plasma GSH/GSSG redox status was positively correlated with the intima thickness of the carotid artery and the oxidized injury of LDL. The redox status shifted to oxidizing direction along with the intima thickening and plaque-forming. Conclusion Elevated peroxidative glutathione redox status was deeply implicated in atherosclerosis progressing, and it may be a sensitive and reliable index for monitoring oxidative status in atherosclerosis.

Effect of atorvastatin on uptake of ox-LDL and expression of CD36 antigen during the transformation from U937cell into foam cell

Background The expression degree of CD36 in monocytes-macrophages is one of the important factors affecting lipid accumulation and foam cell transformation. Atorvastatin has anti-atherosclerosis as well as lowering blood lipid. Thus, we investigate the effect of atorvastatin on expression of CD36 and uptake of oxidized low-density lipoprotein（ox-LDL） during the formation of macrophage-derived foam cells human U937 cell line. Methods U937 cells were incubated with ox-LDL 80 mg / L to induce their transformation into foam cells. The medium was pretreated with atorvastatin 10 nmol / L. The contents of total cholesterol（TC） and cholesterol ester（CE） in cells were measured by the enzymatic fluorometric method. CD36 protein and mRNA expression levels were measured by flow cytometry and reverse transcription PCR. Results After incubated with ox-LDL, the contents of TC and CE in U937 cells increased from 302 mg / g cell protein and87 mg / g cell protein to 469 mg / g cell protein and 226 mg / g cell protein respectively. CD36 protein and mRNA expression appeared. Incubated together with atorvastatin and ox-LDL, the contents of TC and CE decreased from 469 mg / g cell protein and 226 mg / g cell protein to 378 mg / g cell protein and 119 mg / g cell protein, the levels of CD36 protein and mRNA also decreased respectively from 25.8% and 1.27 to 17.2% and0.95 compared with being incubated only with ox-LDL. Conclusion Atorvastatin could inhibit the expression of CD36 protein and mRNA in U937 cells and decrease lipid deposition, which is the important mechanism of anti-atherosclerosis as well as lowering blood lipid.

The relationship between LDL oxidation and macrophage myeloperoxidase activity

Objective To explore low density lipoprotein (LDL) oxidation by macrophage myeloperoxidase (MPO) at molecular level.Methods Using a mouse macrophage model, we examined the relationship between LDL oxidation and macrophage MPO by measuring macrophage MPO activity, LDL oxidation products, MPO gene expression and cellular orientation of LDL oxidation. Results MPO gene expression increased to its maximum gradually when the concentration of LDL was increased, and then maintained at that level. NaN 3 inhibied the elevation of MPO activity and LDL oxidation, which was LDL concentration-dependent. After the composition of macrophage membrane was roughly analyzed, it was determined that the contents of MPO and LDL in 5% sucrose were 7.667 and 21 times higher than those in 10% sucrose, respectively. Conclusion LDL is attached to the “microdomain” of the macrophage membrane in which LDL is oxidized by MPO.