Post-transcriptional control of Amblyomin-X on secretion of vascular endothelial growth factor and expression of adhesion molecules in endothelial cells.

Angiogenesis is a pivotal process of homeostasis and tissue repair, but it also favours neovascularisation syndromes and cancer nutrition. The chemical mediation of angiogenesis is complex, involving a balance between serine proteases and their inhibitors. We addressed the mechanisms of action of a Kunitz serine protease inhibitor (KPI) on spontaneous angiogenesis, using Amblyomin-X, a KPI designed from the cDNA library of the Amblyomma cajennense tick. Amblyomin-X treatment (10-1000 ng/10 µL; each 48 h; 3 times) reduced the number of vessels in the subcutaneous dorsal tissue of male Swiss mice, as measured by intravital microscopy, haematoxylin-eosin staining, and PECAM-1 immunofluorescence labeling. Incubation of Amblyomin-X with t-End endothelial cells, a murine endothelial microvascular lineage, did not alter cell proliferation, cell-cycle phases, necrosis and apoptosis, and the production of nitric oxide and prostaglandin E2. Nevertheless, Amblyomin-X treatment reduced t-End migration and adhesion to Matrigel(®), and inhibited the VEGF-A secretion and VCAM-1 and ß3 integrin expressions by posttranscriptional pathways. Together, data herein outline novel posttranscriptional mechanisms of KPIs on endothelial cells during angiogenesis and point out the possible application of Amblyomin-X as a local inhibitor to undesired neovascularisation process.

Anti-inflammatory and antioxidant properties of a new arylidene-thiazolidinedione in macrophages.

Thiazolidinediones (TZDs) are a class of drugs used for treatment of type 2 diabetes. However, the therapy with currently available TDZs (e.g. rosiglitazone) is associated with important side effects, such as edema and weight gain, suggesting that the investigation of alternative TZDs with better pharmacological properties is warranted. In this study, we investigated both anti-inflammatory and antioxidant properties of a new chemically modified TZD, the arylidene-thiazolidinedione 5-(4-methanesulfonyl-benzylidene)-3-(4-nitrobenzyl)-thiazolidine-2,4-dione (SF23), and compared the results to those obtained with rosiglitazone. We found that our SF23 displays a weaker affinity for PPARγ, up-regulating in a lower magnitude the expression of both PPARγ and CD36 compared to rosiglitazone. In lipopolysaccharide (LPS)-stimulated macrophages, SF23 decreased nitrite production and attenuated the mRNA expression of both iNOS and COX-2. These anti-inflammatory effects were comparable to those obtained with rosiglitazone. Interestingly, SF23, but not rosiglitazone, prevented LPS-induced mitochondrial membrane hyperpolarization, apoptosis, reactive oxygen species (ROS) generation, and the expression of NADPH oxidase subunits, Nox1 and Nox2. In addition, in macrophages from Nrf2⁻/⁻ mice, SF23 protected against LPSinduced cellular death and ROS production, whereas rosiglitazone was only able to protect normal Nrf2⁺/⁺ cells against oxidative injury, suggesting that, unlike rosiglitazone, the antioxidant activity of SF23 might be Nrf2-independent. Finally, in macrophages exposed to high concentrations of glucose, SF23 induced significant increases in the mRNA expression of glucose transporters, insulin receptor substrate and mitoNEET. Altogether, our data indicate that our new chemically modified TDZ displays similar anti-inflammatory properties, but superior antioxidant effects on the LPS-stimulated macrophages compared to rosiglitazone.

Cu and Fe metallic ions-mediated oxidation of low-density lipoproteins studied by NMR, TEM and Z-scan technique.

In this work we report on a study of the morphological changes of LDL induced in vitro by metallic ions (Cu(2+) and Fe(3+)). These modifications were characterized by transmission electron microscopy, nuclear magnetic resonance and the Z-scan technique. The degree of oxidative modification of LDL was determined by the TBARS and lipid hydroperoxides assays. It is shown that distinct pathways for modifying lipoproteins lead to different morphological transformations of the particles characterized by changes in size and/or shape of the resulting particles, and by the tendency to induce aggregation of the particles. There were no evidence of melting of particles promoted by oxidative processes with Cu and Fe.

Electronegative LDL induction of apoptosis in macrophages: involvement of Nrf2.

The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(-)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264.7 macrophages with LDL(-) for 24 h resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(-); incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(-)-treated cells. CD95 (Fas), CD95 ligand (FasL), CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(-)-treated cells. However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(-) treatment. LDL(-) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL. Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(-), together with an increase in the production of ROS in the absence of alterations in CD36 expression. These results provide evidence that CD36 expression induced by LDL(-) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(-)-induced apoptosis in macrophages.

Cu and Fe metallic ions-mediated oxidation of low-density lipoproteins studied by NMR, TEM and Z-scan technique

In this work we report on a study of the morphological changes of LDL induced in vitro by metallic ions (Cu2+ and Fe3+). These modifications were characterized by transmission electron microscopy, nuclear magnetic resonance and the Z-scan technique. The degree of oxidative modification of LDL was determined by the TBARS and lipid hydroperoxides assays. It is shown that distinct pathways for modifying lipoproteins lead to different morphological transformations of the particles characterized by changes in size and/or shape of the resulting particles, and by the tendency to induce aggregation of the particles. There were no evidence of melting of particles promoted by oxidative processes with Cu and Fe.

Antibodies against electronegative LDL inhibit atherosclerosis in LDLr-/- mice

In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 µg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25 percent cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm² (mean ± SD), respectively) compared to control (124.9 ± 13.2 µm²). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 ± 0.70 per field x 10) compared to controls (21.5 ± 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 ± 1.4; control: 20.5 ± 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 ± 2.7; control: 15.0 ± 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 ± 1.5; control: 30.0 ± 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 ± 0.020; control: 0.38 ± 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

Electronegative LDL and lipid abnormalities in patients undergoing hemodialysis and peritoneal dialysis.

BACKGROUND: Oxidative modification of low-density lipoprotein (LDL) has been demonstrated in patients with end-stage renal disease, where it is associated with oxidative stress and plays a key role in the pathogenesis of atherosclerosis. In this context, the generation of minimally oxidized LDL, also called electronegative LDL [LDL(-)], has been associated with active disease, and is a detectable sign of atherogenic tendencies. The purpose of this study was to evaluate serum LDL(-) levels and anti-LDL(-) IgG autoantibodies in end-stage renal disease patients on dialysis, comparing patients on hemodialysis (HD), peritoneal dialysis (PD) and a control group. In addition, the serum lipid profile, nutritional status, biochemical data and parameters of mineral metabolism were also evaluated. METHODS: The serum levels of LDL(-) and anti-LDL(-) IgG autoantibodies were measured in 25 patients undergoing HD and 11 patients undergoing PD at the Centro Integrado de Nefrologia, Rio de Janeiro, Brazil. Ten healthy subjects served as a control group. Serum levels of albumin, total cholesterol, triglycerides and lipoproteins were measured. Calculations of subjects' body mass index and measurements of waist circumference, triceps skin fold and arm muscle area were performed. Measurements of hematocrit, serum blood urea nitrogen, creatinine, parathyroid hormone, phosphorus and calcium were taken. RESULTS: Levels of LDL(-) were higher in HD patients (575.6 +/- 233.1 microg/ml) as compared to PD patients (223.4 +/- 117.5 microg/ml, p < 0.05), which in turn were higher than in the control group (54.9 +/- 33.3 mug/ml, p < 0.01). The anti-LDL(-) IgG autoantibodies were increased in controls (0.36 +/- 0.09 microg/ml) as compared to PD (0.28 +/- 0.12 microg/ml, p < 0.001) and HD patients (0.2 +/- 0.1 microg/ml, p < 0.001). The mean values of total cholesterol and LDL were considered high in the PD group, whereas the mean triceps skin fold was significantly lower in the HD group. CONCLUSION: Levels of LDL(-) are higher in renal patients on dialysis than in normal individuals, and are reciprocally related to IgG autoantibodies. LDL(-) may be a useful marker of oxidative stress, and this study suggests that HD patients are more susceptible to cardiovascular risk due to this condition. Moreover, autoantibodies reactive to LDL(-) may have protective effects in chronic kidney disease.

Antibodies against electronegative LDL inhibit atherosclerosis in LDLr-/- mice.

In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 microg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25% cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 +/- 9.7, 67.3 +/- 17.02, 56.9 +/- 8.02 microm(2) (mean +/- SD), respectively) compared to control (124.9 +/- 13.2 microm(2)). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 +/- 0.70 per field x 10) compared to controls (21.5 +/- 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 +/- 1.4; control: 20.5 +/- 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 +/- 2.7; control: 15.0 +/- 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 +/- 1.5; control: 30.0 +/- 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 +/- 0.020; control: 0.38 +/- 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

Cholesterol oxides as biomarkers of oxidative stress in type 1 and type 2 diabetes mellitus.

BACKGROUND: Oxidative stress plays an important role in the pathophysiology of diabetes mellitus. The aim of this study was to evaluate the formation of cholesterol oxides (ChOx) as biomarkers of oxidative stress in subjects with impaired glucose tolerance (IGT) and diabetes. METHODS: Blood plasma levels of cholesterol oxidation products were determined in the following groups: type 1 diabetes mellitus (DM1), type 2 diabetes (DM2), impaired glucose tolerance (IGT), children without diabetes (C1) and adults without diabetes (C2). The serum levels of cholest-5-ene-3alpha,7alpha-diol (7alpha-hydroxycholesterol, 7alpha-OH), cholest-5-ene-3beta,7beta-diol (7beta-hydroxycholesterol, 7beta-OH), 3beta-hydroxycholest-5-7-one (7-ketocholesterol, 7-K), 5alpha-cholestane-3beta,5,6beta-triol (cholestanetriol), 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol (cholesterol-5alpha,6alpha-epoxide,), 5,6beta-epoxy-5beta-cholestan-3beta-ol (cholesterol-5beta,6beta-epoxide) and cholest-5-eno-3beta,25-diol (25-hydroxycholesterol, 25-OH) (trivial name and abbreviations indicated in parentheses) were quantified by gas chromatography using flame ionization detection. RESULTS: The levels of total ChOx were elevated in the DM1 and DM2 groups compared to age-matched subjects without diabetes (p < 0.05). The concentrations of 7beta-hydroxycholesterol, cholesterol-alpha-epoxide and cholesterol-beta-epoxide were higher in the blood plasma of subjects in the DM2 group than in the blood plasma of subjects in the C2 and IGT groups (p < 0.05). Treatment of type 2 diabetic patients with oral hypoglycemic drugs associated with insulin resulted in lower concentrations of nitrotyrosine in the blood plasma without significant changes in the concentrations of glucose and glycated hemoglobin. Moreover, combination with statins in both treatments decreased the concentrations of ChOx. CONCLUSIONS: ChOx are suitable biomarkers of oxidative stress and may be useful in clinical studies to follow drug effects on lipid oxidative modifications in diabetic patients.

Cholesterol oxides as biomarkers of oxidative stress in type 1 and type 2 diabetes mellitus / Cholesterol oxides as biomarkers of oxidative stress in type 1 and type 2 diabetes mellitus

Background Oxidative stress plays an important role in the pathophysiology of diabetes mellitus. The aim of this study was to evaluate the formation of cholesterol oxides (ChOx) as biomarkers of oxidative stress in subjects with impaired glucose tolerance (IGT) and diabetes. Methods Blood plasma levels of cholesterol oxidation products were determined in the following groups: type 1 diabetes mellitus (DM1), type 2 diabetes (DM2), impaired glucose tolerance (IGT), children without diabetes (C1) and adults without diabetes (C2). The serum levels of cholest-5-ene-3£/,7£/-diol (7£/-hydroxycholesterol, 7£/-OH), cholest- 5-ene-3£],7£]-diol (7£]-hydroxycholesterol, 7£]-OH), 3£]-hydroxycholest-5-7- one (7-ketocholesterol, 7-K), 5£/-cholestane-3£],5,6£]-triol (cholestanetriol), 5,6£/-epoxy-5£/-cholestan-3£/-ol (cholesterol-5£/,6£/-epoxide,), 5,6£]-epoxy- 5£]-cholestan-3£]-ol (cholesterol-5£],6£]-epoxide) and cholest-5-eno-3£],25-diol (25-hydroxycholesterol, 25-OH) (trivial name and abbreviations indicated in parentheses) were quantified by gas chromatography using flame ionization detection. Results The levels of total ChOx were elevated in the DM1 and DM2 groups compared to age-matched subjects without diabetes (p < 0.05).The concentrations of 7£]-hydroxycholesterol, cholesterol-£/-epoxide and cholesterol-£]-epoxide were higher in the blood plasma of subjects in the DM2 group than in the blood plasma of subjects in the C2 and IGT groups (p < 0.05). Treatment of type 2 diabetic patients with oral hypoglycemic drugs associated with insulin resulted in lower concentrations of nitrotyrosine in the blood plasma without significant changes in the concentrations of glucose and glycated hemoglobin. Moreover, combination with statins in both treatments decreased the concentrations of ChOx.Conclusions ChOx are suitable biomarkers of oxidative stress and may be useful in clinical studies to follow drug effects on lipid oxidative modifications in diabetic patients. Copyright ÆÉ 2006 John Wiley & Sons, Ltd.

Nitric oxide, cholesterol oxides and endothelium-dependent vasodilation in plasma of patients with essential hypertension.

The objective of the present study was to identify disturbances of nitric oxide radical (.NO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations of.NO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 +/- 9.7 years; blood pressure, 148.3 +/- 24.8/90.8 +/- 10.2 mmHg) and in 11 healthy subjects (N: 48.4 +/- 7.0 years; blood pressure, 119.4 +/- 9.4/75.0 +/- 8.0 mmHg). Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1% increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 +/- 26.0, N: 54.2 +/- 24.9 micro M), urate (H: 108.5 +/- 18.9, N: 156.4 +/- 26.3 micro M), beta-carotene (H: 1.1 +/- 0.8, N: 2.5 +/- 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 +/- 0.2, N: 0.7 +/- 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3beta,5,6beta-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 +/- 0.2, N: 0.7 +/- 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for.NO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although.NO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides.

Nitric oxide, cholesterol oxides and endothelium-dependent vasodilation in plasma of patients with essential hypertension

The objective of the present study was to identify disturbances of nitric oxide radical (ANO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations ofANO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 ± 9.7 years; blood pressure, 148.3 ± 24.8/90.8 ± 10.2 mmHg) and in 11 healthy subjects (N: 48.4 ± 7.0 years; blood pressure, 119.4 ± 9.4/75.0 ± 8.0 mmHg).Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1 percent increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 ± 26.0, N: 54.2 ± 24.9 æM), urate (H: 108.5 ± 18.9, N: 156.4 ± 26.3 æM), ß-carotene (H: 1.1 ± 0.8, N: 2.5 ± 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 ± 0.2, N: 0.7 ± 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3ß,5,6ß-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 ± 0.2, N: 0.7 ± 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for ANO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although ANO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides

Biomarcador da modificaçäo oxidativa da LDL in vivo / Biomarker LDL's oxidative modification in vivo

As modificaçöes oxidativas da lipoproteína de baixa densidade (LDL) säo consideradas um fator importante para o desenvolvimento da aterosclerose. Estas modificaçöes ocorrem in vivo, originando uma sub-traçäo denominada de LDL, eletronegativa (LDL-). O monitoramento clínico da LDL- é de extrema importância, mas estava sendo limitado pela dificuldade para detecçäo desta partícula em fluídos biológicos. Neste estudo desenvolveu-se novas metodologias para detectar a LDL- no plasma, utilizando-se um anticorpo monoclonal anti-LDL- humana (3D1036) e avaliar a resposta imune humoral relacionada à LDL-. A LDL- plasmática foi analisada através de um ELISA com detecçäo por quimioluminescência com boa sensibilidade (<1,0µg/mL) e precisäo (CVintra=6,44 ñ 1,15 porcento e CVinter=8,59 ñ 3,42 porcento). As análises dos auto-anticorpos anti-LDL- evidenciaram a presença de uma resposta imune específica para LDL- em humanos e em coelhos. A determinaçäo da LDL-, abre novas perspectivas para o monitoramento das modificaçöes oxidativas endógenas da LDL em estudos clínicos e de intervençäo que utilizam um elevado número de amostras. Além disto, a detecçäo dos auto-anticorpos anti-LDL- demonstra o potencial imunogênico desta partícula. Portanto, a detecçäo da LDL- e dos auto-anticorpos anti-LDL- abre novas perspectivas para o monitoramento dos fatores de risco para a aterosclerose vinculados às reaçöes oxidativas

Plasma clearance and biodistribution of oxidatively modified 99mTc-Beta-VLDL in rabbits

The biodistribution and removal from plasma (measured as fractional clerance rate, FCR, per hour) of native and oxidatively modified (99m)technetium-labeled Beta-very low density lipoprotein ((99m)Tc-Beta-VLDL)) were investigated in hypercholesterolemic (HC) and control (C) three-month old New Zealand rabbits. The intracellular accumulation of Beta-VLDL labeled with (99m)Tc was studied in vitro in THP-1 cells and monocyte-derived macrophages isolated from rabbits. After intravenous injection into C rabbits, copper-oxidized Beta-VLDL ((99m)Tc-ox-Beta-VLDL)) was cleared from the circulation faster (0.362 + 0.070/h) than native Beta-VLDL ((99m)Tc-nat-Beta-VLDL, 0.241 + 0.070/h)). In contrast, the FCR of (99m)Tc-ox-Beta-VLDL in HC rabbits was lower (0.100 + 0.048/h) than that of (99m)Tc-nat-Beta-VLDL (0.163 + 0.043/h). The hepatic uptake of radiolabeled lipoproteins was lower in HC rabbits (0.114 + 0.071 percent injected dose/g tissue for (99m)Tc-nat-Beta-VLDL and 0.116 + 0.057 percent injected dose/g tissue for (99m) Tc-ox-Beta-VLDL) than in C rabbits (0.301 + 0.113 percent injected dose/g tissue for (99m)Tc-nat-Beta-VLDL and 0.305 + 0.149 percent injected dose/g tissue for ((99m)Tc-ox-Beta-VLDL). The uptake of (99m)Tc-nat-Beta-VLDL and (99m)Tc-ox-Beta-VLDL by atherosclerotic aorta lesions isolated from HC rabbits ((99m)Tc-nat-Beta-VLDL:0.033 + 0.012 percent injected dose/g tissue and (99m)Tc-ox-Beta-VLDL: 0.039 + 0.017 percent injected dose/g tissue) was higher in comparison to that of non-atherosclerotic aortas from C rabbits (99m)Tc-nat-Beta-VLDL: 0.023 + 0.010 percent injected dose/g tissue and (99m)Tc-ox-Beta VLDL: 0.019 + 0.010 percent injected dose/g tissue). However, (99m) Tc-nat-Beta-VLDL and (99m)Tc-ox-Beta-VLDL were taken up by atherosclerotic lesions at similar rates. In vitro studies showed that both monocyte-derived macrophages isolated from rabbits and THP-1 macrophages significantly internalized more (99m)Tc-ox-Beta-VLDL than (99m)Tc-nat-Beta-VLDL. These results indicate that in cholesterol-fed rabbits (99m)Tc-ox-Beta-VLDL is slowly cleared from plasma and accumulates in atherosclerotic lesions. However, although the extent of in vitro uptake of (99m)Tc-ox-Beta-VLDL by macrophages was high, the in vivo accumulation of this radiolabeled lipoprotein by atherosclerotic lesions did not differ from that of (99m)Tc-nat-Beta-VLDL.

Monoclonal antibodies against low density lipoprotein with various degrees of oxidative modifications

Oxidative processes leading to the generation of oxidized low density lipoprotein (oxLDL) particles have been suggested to be an important factor in the pathogenesis of atherosclerosis. After initiation of the oxidative process, LDL undergoes a progressive protein and lipid fragmentation. To understand this process and the role of oxLDL in various diseases of inflammatory origin, we have generated mouse monoclonal antibodies against copper-oxidized human LDL. Mice were immunized intrasplenically and after one intravenous boost the spleen cells were fused with the Sp2/0 hybridoma fusion partner. The hybridoma clones obtained after selection and cloning were analyzed for reactivity against oxLDL with various degrees of copper-mediated oxidative modifications. Three hybridoma clones were purified and further characterized. The following observations were made: 1) the intrasplenic route of immunization, avoiding the use of mycobacterial adjuvants, yielded a high frequency of positive clones; 2) the individual hybridomas reacted against LDL with various degrees of oxidative modifications; 3) the monoclonal antibodies could be used in ELISA and to detect oxLDL in immunohistochemical tissue staining, and 4) the monoclonal antibodies also detected oxLDL from hamsters and rabbits. We conclude that these monoclonal antibodies could be useful to further investigate the role of oxLDL in inflammation and in the immune response.