Troglitazone-binding to LDL and its glycated modifications: its role in cell-catalysed and Cu-mediated LDL-oxidation

Troglitazone (T), an anti-diabetic drug improving insulin resistance, was studied as to its inhibition of copper ion-catalysed oxidation of native, glycated and glycoxidated low-density lipoprotein (LDL). A dose-dependent inhibition was noted in the concentration range 40-160 microg/ml. An almost complete inhibition of oxidation (2-8 h), as monitored by the formation of thiobarbituric acid-reactive substances, was observed for both native and glycated LDL at a concentration of 160 microg/ml T, while the maximal inhibition for glycoxidated LDL amounted only to 60% at this concentration of the drug. This is reflected by differences in the affinity of the drug for the different types of LDL modification: While the binding of T both to native or glycated LDL increased linearly with increasing T concentration and was not saturable in the concentration range tested (0-160 microg/ml), binding of the drug to glycoxidated LDL was already nearly saturated at 10 microg/ml. The nearly complete inhibitory action of T towards oxidation of native and glycated LDL was lost, however, upon increasing the total oxidation time to 24 h. In human umbilical vein endothelial cell-mediated oxidation of LDL, T at a concentration of 20 microg/ml significantly reduced formation of oxidation-dependent fluorescent chromophores and liberation of 8-epi-PGF2alpha. In contrast, generation of thiobarbituric acid-reactive substances was not significantly inhibited. As opposed to copper-mediated LDL-oxidation, different binding of T to LDL-modifications does not govern inhibition of human umbilical vein endothelial cell-mediated LDL-oxidation.     

Influence of acetylsalicylic acid on oxidation of native and glycated low-density lipoprotein

It is generally accepted that oxidation of low-density lipoproteins (LDL) is a causal factor in the development of atherosclerosis. Non-enzymatic glycosylation of LDL, i.e."glycation", plays a central role in late complications of diabetes mellitus and may initiate and/or accelerate the oxidation process. Therefore, the inhibition of this processes is of major therapeutic relevance. The influence of acetylsalicylic acid (ASA) on the oxidation of native and glycated LDL was studied in vitro. LDL (0.25 mg protein/ml ) was oxidatively modified with 5.0 microM CuSO4. Only at "supratherapeutical" ASA concentrations in the range 0.06-2.0 mg /ml we found a significant concentration-dependent inhibition of LDL oxidation both for native and glycated LDL, which was from 0.2 mg/ml upwards significantly more marked for native LDL than for glycated LDL. The maximal inhibitory effect occurred at 2.0 mg/ml with 89.6% inhibition of LDL-oxidation for native LDL and 64.4% for glycated LDL. At 0.2 mg/ml ASA the respective inhibitory values were 38.5% and 31.0%. For glycated LDL the ASA doses of maximal- and approximately 50%-inhibition, as found for native LDL, were chosen to investigate the inhibitory effect on 2,4,8 and 24 hours oxidation of glycated LDL to monitor the time-dependency of inhibition by ASA. This revealed that ASA only delayed, not permanently inhibited LDL oxidation.     

Oxidized low density lipoprotein inhibits prostacyclin generation by rat aorta in vitro: a key role of lysolecithin.

The objective of this study was to examine the effect of oxLDL on prostacyclin (PGI2) generation by rat aortic segments and to see whether the lipid fraction of oxLDL or its components are responsible for that effect. We also tested if antioxidants have any protective role. LDL oxidized by copper was characterized by higher TBARS, conjugated diene, lysophosphatidylcholine (lyso PC), oxysterols and less polyunsaturated fatty acids (PUFA) than nLDL. Preincubation of aortas with oxLDL caused a significant inhibition of PGI2 generation compared to aortas preincubated with nLDL or buffer only. The percent inhibition was dependent on the concentration of oxLDL. Most of the inhibitory effect of oxLDL resided in its lipid moiety while the lipid fraction of nLDL, as well as native LDL had no effect. Preincubation of aortas with 10 microg/ml of 7-ketocholesterol the major oxysterol in oxLDL reduced the amount of PGI2 generated by aorta at all times tested; however that decrease did not reach a significant level. Aortas preincubated with 10 microg/ml of lyso PC showed a 21-36% inhibition of PGI2 generation which was comparable to the inhibition produced by preincubating the aortas with 50 microg protein/ml of oxLDL (containing about 7.5 microg lyso PC). This indicated that most of the inhibitory effect of oxLDL was due to its lyso PC. The small molecular weight fraction (< 10 kDa) with a high level of TBARS (TBARS solution) also significantly decreased the PGI2 generation by aorta. Addition of superoxide dismutase (SOD) + catalase or vitamin E simultaneously with oxLDL or TBARS solution in the preincubation medium did not reverse their inhibitory effects. This indicated that oxygen free radicals are not a contributing factor to the inhibitory effect of oxLDL but lyso PC and the lipid peroxides and probably other components already present within oxLDL are the important inhibitors.     

The effects of glycation/glycoxidation on the liberation of 8-epi-PGF2alpha from low density lipoprotein during its in vitro oxidation

In individuals suffering from diabetes mellitus, low-density lipoprotein (LDL) can undergo glycoxidation, both the surface protein and the unsaturated fatty acids in the particle core experiencing oxidative damage. In these patients, plasma levels of glycated(g)/glycoxidated(go) low-density lipoprotein and of 8-epi-PGF2alpha (8-IP), a relatively stable peroxidation product of arachidonic acid, are increased. This study reports on the modified oxidation of gLDL and goLDL by human umbilical vein endothelial cells or by copper cations in the absence of cells. In both systems, glycated LDL was found to be more easily oxidized than either nLDL or goLDL. In addition, liberation of 8-IP from glycoxidated LDL is significantly reduced, because a large amount of 8-IP is already formed during long-term glycation of LDL in the absence of metal chelators, i.e. during glycoxidation. From these in vitro results we conclude, that 1) gLDL is more prone to cell-mediated oxidation than native or goLDL and 2) the increased in vivo plasma levels of 8-IP observed in diabetes mellitus could be due to prolonged LDL-glycoxidation liberating continuously significant amounts of 8-IP.     

Ibuprofen protects low density lipoproteins against oxidative modification

Oxidative modification of LDL by vascular cells has been proposed as the mechanism by which LDL become atherogenic. The effect of ibuprofen on LDL modification by copper ions, monocytes and endothelial cells was studied by measuring lipid peroxidation products. Ibuprofen inhibited LDL oxidation in a dose-dependent manner over a concentration range of 0.1 to 2.0 mM. Ibuprofen (2 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during 2 and 6 h incubation in the presence of copper ions by 52 and 28%, respectively. Weak free radical scavenging activity of ibuprofen was observed in the DPPH test. The protective effect of ibuprofen was more marked when oxidation was induced by monocytes or endothelial cells. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides generated in LDL during monocyte-mediated oxidation by 40%. HUVEC-mediated oxidation of LDL in the absence and presence of Cu2+ was reduced by 32 and 39%, respectively. More lipid peroxides appeared when endothelial cells were stimulated by IL-1beta or TNFalpha and the inhibitory effect of ibuprofen in this case was more pronounced. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during incubation of LDL with IL-1beta-stimulated HUVEC by 43%. The figures in the absence and presence of Cu2+ for HUVEC stimulated with TNFalpha were 56 and 59%, respectively. To assess the possibility that ibuprofen acts by lowering the production rate of reactive oxygen species, the intracellular concentration of H2O2 was measured. Ibuprofen (1 mM) reduced intracellular production of hydrogen peroxide in PMA-stimulated mononuclear cells by 69%. When HUVEC were stimulated by IL-1beta or TNFalpha the reduction was 62% and 66%, respectively.     

High density lipoprotein can modulate the inhibitory effect of oxLDL on prostacyclin generation by rat aorta in vitro

To examine the effect of oxidized low density lipoprotein (oxLDL) on prostacyclin (PGI2) generation by rat aorta in vitro and whether high density lipoprotein (HDL) has any protective effect against the inhibition of PGI2 generation induced by oxLDL is the objective of this study. Preincubation of aortas with oxLDL resulted in significant inhibition of PGI2 generation compared to preincubation with normal low density lipoprotein (nLDL) or buffer only. The inhibitory effect of oxLDL resided in its lipid moiety while the lipid fraction of nLDL showed no effect. Aortas preincubated with 10 microg/ml of lyso phosphatidycholine (lyso PC) also showed 30% inhibition of PGI2 generation, indicating that lyso PC was among the lipid components of oxLDL which inhibited PGI2 generation. Preincubation of aortas with a mixture of HDL and oxLDL at a ratio of 10:1 showed a significant recovery of PGI2 generation compared to aortas preincubated with only oxLDL, indicating a protective role for HDL. When HDL was incubated with oxLDL the transfer of lyso PC from oxLDL to HDL suggested that HDL trapped lyso PC from oxLDL thus preventing it from acting on the aorta. However, when a mixture of HDL and oxLDL at a ratio of 3:1 was preincubated with aortas, no protective effect of HDL was observed. Preincubation of aortas with a mixture of HDL plus oxLDL at a ratio of 8:1, which was incubated for 1 h at 37 degrees C, produced significantly less PGI2 than aortas preincubated only with oxLDL, indicating that HDL under these conditions was not protective but even enhanced the inhibitory effect of oxLDL. Similarly, aortas preincubated with HDL plus whole oxLDL (at a ratio of 10:1); containing all the small molecular weight oxidation products and characterized by high levels of thiobarbituric acid reactive substance (TBARS) and lipid hydroperoxides; produced significantly less PGI2 than aortas preincubated with whole oxLDL. These results were evaluated in light of possible modification of HDL by oxLDL and its lipid oxidation products such as aldehydes and lipid peroxides. The modified HDL can add more lipid peroxides and increase the effectiveness of lipid peroxides originally present in oxLDL.     

Comparison of Iloprost, Cicaprost and prostacyclin effects on cyclic AMP metabolism in intact platelets.

We have compared the effects of prostacyclin (PGI2) and its stable analogs, Iloprost and Cicaprost, on cyclic AMP metabolism in intact platelets. All three compounds show similar but not identical patterns of prostaglandin concentration-dependent cyclic AMP formation. All three compounds apparently stimulate and inhibit cyclic AMP formation with different concentration dependencies, indicating the presence of distinct stimulatory and inhibitory receptors. Differences in response can be accounted for by slight differences in affinity of stimulatory and inhibitory receptors for the prostaglandins, by the fact that Iloprost contains almost 50% of a relatively inactive isomer, and by the fact that PGI2 is labile in aqueous solution, with a half-life on the order of a few minutes. We conclude 1) stimulation and inhibition of adenylate cyclase is not due to separate effects of 16S- and 16R-stereoisomers of Iloprost because similar patterns were obtained with a single isomeric form of Cicaprost and with authentic PGI2; 2) prostaglandin induced inhibition of adenylate cyclase is readily reversible because inhibition disappears when PGI2 concentration decays below saturation of the inhibitory receptor; 3) the potency of prostaglandins in stimulating platelet adenylate cyclase must be viewed in terms of their effects on both stimulatory and inhibitory receptors.     

Short peptide receptor mimics for atherosclerosis risk assessment of LDL

Short peptides sequences were selected that showed binding selectivity towards healthy or oxidised (unhealthy) low density lipoprotein (LDL), respectively. These were investigated for application in atherosclerosis risk monitoring. Comparison was also made with the LDL receptor ligand repeat peptide (LR5). The peptides were immobilised on a gold surface plasmon resonance surface and LDL binding detected as a shift in the resonance. 3.7x10(7) (+/-5.6x10(6)) LDL/mm(2)/microg/ml solution LDL were bound on GlySerAspGlu-OH and 6.8x10(7) (+/-9.2x10(6)) LDL/mm(2)/microg/ml on GlyCystineSerAspGlu, compared with approximately 10(8) LDL/mm(2)/microg/ml on LR5. In this first group, binding of LDL decreased with oxidation level and a good correlation was found between LDL binding and residual amino groups on the apoprotein of the LDL following oxidation, or the change in relative electrophoretic mobility (REM) of LDL. The decrease in binding was 1.1x10(7) LDL particles/mm(2) per% oxidation for GlySerAspGlu-OH, 1.8x10(7) LDL particles/mm(2) per% oxidation for GlyCystineSerAspGlu and 2.4x10(7) LDL particles/mm(2) per% oxidation for LR5. A second group of three peptides were also selected showing increased binding with LDL oxidation: GlyCystineCysCys (1.5x10(7) LDL/mm(2) per microg/ml), GlyLysLysCys-SH (10(7) LDL/mm(2) per microg/ml) and GlyLysLys-OH (5.6x10(7) LDL/mm(2) per microg/ml). The latter gave a linear increase in LDL binding with oxidation level (1.2x10(7) LDL particles/mm(2) per% oxidation). LDL concentration is around 2-3 mg/ml in plasma compared with the low detection levels with this method (1-10 microg/ml), allowing a strategy to be developed requiring the minimum sample volume and diluting with physiological buffer prior to assay. By using a comparative reading between LDL adsorption on surfaces from the first and second group of peptides (e.g. GlyCystineSerAspGlu and GlyLysLys-OH, respectively), LDL oxidation could be determined without knowledge of LDL concentration. Higher binding was seen on GlyCystineSerAspGlu than GlyLysLys-OH below 30% LDL oxidation, whereas above 30% oxidation the binding on the latter surface was greater. Simple correlation of this form could provide good tests for atherosclerosis risk.     

Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and of incorporation of acetate into cholesterol in homozygous hypercholesterolemic fibroblasts by ferritin-low density lipoprotein conjugates.

Conjugates of ferritin with low density lipoproteins (LDL) were prepared and separated by sucrose gradient centrifugation. These conjugates, at cholesterol concentration of 100--132 microgram/ml, caused a greater than 90% suppression of hydroxymethylglutaryl coenzyme A reductase activity and of acetate incorporation into cholesterol in cultured skin fibroblasts from a normal subject as well as from a subject with homozygous familial hypercholesterolemia. The half maximal inhibition concentration was approx. 10 microgram/ml cholesterol for LDL and ferritin . (LDL)2 and 5 microgram/ml for (ferritin)2 . LDL in both cell lines. In contrast, native low density lipoproteins have only a minimal inhibitory effect in homozygous cells. The ability of the conjugates to stimulate the incorporation of oleate into cholesteryl esters was also equal in the two cell lines, although the conjugates were only 10% as active as low density lipoproteins in the normal cells. LDL reduced the ferritin . (LDL)2-mediated suppression of hydroxymethylglutaryl-CoA reductase activity in homozygous cells while ferritin . (LDL)2 reduced the LDL-mediated stimulation of cholesteryl ester formation in normal cells.     

Three different pathways for human LDL oxidation are inhibited in vitro by water extracts of the medicinal herb Achyrocline satureoides

In this study we investigated the antioxidant properties of one herbal preparation widely used in complementary and alternative medicine in large areas of the world: Achyrocline satureoides (AS), popularly known as "marcela". Although rich in flavonoids, the ethnopharmacological uses of this plant do not include atherosclerosis prevention. Furthermore, no study had been conducted so far exploring the antioxidant activity of Achyrocline satureoides vis-à-vis human LDL oxidation, which is the compelling issue in pinpointing potential cardioprotective new uses for a traditional remedy. We explored the effects of AS extracts on human LDL oxidation, employing 3 different systems which are thought to play a role in oxidation of LDL in the arterial wall: copper, peroxynitrite, and lipoxygenase. Oxidation was monitored by conjugate dienes, TBARS formation and aggregation of apoB using SDS-PAGE. In copper-initiated oxidation a dose dependent inhibition of the initiation and propagation of lipid oxidation is shown by an increase in the lag phase for conjugate diene production which was 60 +/- 15 min in the absence and 120 +/- 20 min in the presence of 4 microg/ml AS extracts (p < 0.001). TBARS production was reduced by 95% after 3 h incubation at 5 microg/ml. Aggregation of apoB was abolished at the same concentrations. SIN-1 (3-morpholinosydnonimine) produces peroxynitrite via generation of NO and O2-. When LDL was incubated in its presence, a milder oxidation was observed as compared with Cu2+, and AS produced over 70% inhibition. Finally, we show a striking dose-dependent inhibitory effect of lipoxygenase conjugate diene production, which is over 95% at AS concentrations of 5 microg/ml. When compared with other antioxidants, AS effect is greater but in the same order of magnitude than that of ascorbic acid and similar to the popular herbal tea Ilex paraguariensis. In all three systems employed an effect is already substantiated at a concentration of the AS extract of 4 microg/ml, which corresponds to a 1/100 dilution of the preparations usually drunk.     

The PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban synergistically inhibit TXA2-dependent platelet activation

The stable PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban (BM 13177) were investigated for possible synergistic effects on platelet aggregation in human platelet rich plasma in vitro. Iloprost and sulotroban synergistically inhibited U 46619, collagen, and the second wave of ADP-induced platelet aggregation. Iloprost and sulotroban at concentrations showing little or no inhibition alone resulted, in combination, in marked or complete inhibition of U 46619 or collagen induced aggregation. Combination of iloprost 10(-10) M, which had no effect on the concentration-response curve (CRC) to U 46619, with sulotroban 5 x 10(-6) M, which shifted the CRC to U 46619 by a factor of 3 to the right, resulted in a rightward shift of the U 46619 CRC by a factor of 4.5. To attain a 4.5-fold shift with either compound alone, a concentration of 5 x 10(-10) M iloprost or 10(-5) M sulotroban was required. A similar mutual enhancement of inhibitory effects was seen for combinations of the PGI2-analogue cicaprost (ZK 96.480) with sulotroban or the TXA2-receptor antagonist SQ 29548 with iloprost. When the TXA2-dependent part of collagen-induced aggregation was fully inhibited by sulotroban, the concentrations of iloprost necessary for 90% inhibition were reduced by a factor of 2.5 - 3. In the presence of acetylsalicylic acid, the synergistic action of sulotroban and iloprost was reduced and merely additive effects against U 46619-induced platelet aggregation were found, suggesting that the release of endogenous TXA2 plays an important role for the synergistic effect of the two compounds. The combination of a PGI2-analogue and a TXA2-antagonist may lead to a safer and more effective control of platelet activation than with either compound alone.     

The role of antioxidants in the long-term glycation of low density lipoprotein and its Cu2+-catalyzed oxidation

In the present study we investigated the influence of antioxidants such as EDTA, α-tocopherol, troglitazone and acetylsalicylic acid on the long-term-glycation of LDL and its copper ion-catalyzed oxidation. We observed that (a) all antioxidants inhibited AGE-formation, while Amadori product formation was only diminished by extreme concentrations of acetylsalicylic acid, (b) glycated LDL was more susceptible to coppercatalyzed oxidation than unglycated LDL, and (c) the oxidation of native LDL was more dramatically inhibited by the antioxidants than that of glycated LDL. The observed differences may be a consequence of the significantly higher endogenous content in hydroperoxides of glycated LDL as compared to native LDL. Therapeutic implications of these findings regarding vitamin E, which is supposed to slow atherogenesis and the development of microvascular complications in diabetes, are obvious: Vitamin E-monotherapy, while blocking oxidative and AGE-modification of LDL, is unable to inhibit its AP-formation. As a consequence, tocopherol is susceptible to increased consumption by AP-associated radical production in hyperglycemic patients, which could be checked in part by the tocopherol-protecting agent troglitazone and/or by acetylsalicylic acid.     

The role of antioxidants in the long-term glycation of low density lipoprotein and its Cu2+-catalyzed oxidation

In the present study we investigated the influence of antioxidants such as EDTA, alpha-tocopherol, troglitazone and acetylsalicylic acid on the long-term-glycation of LDL and its copper ion-catalyzed oxidation. We observed that (a) all antioxidants inhibited AGE-formation, while Amadori product formation was only diminished by extreme concentrations of acetylsalicylic acid, (b) glycated LDL was more susceptible to copper-catalyzed oxidation than unglycated LDL, and (c) the oxidation of native LDL was more dramatically inhibited by the antioxidants than that of glycated LDL. The observed differences may be a consequence of the significantly higher endogenous content in hydroperoxides of glycated LDL as compared to native LDL. Therapeutic implications of these findings regarding vitamin E, which is supposed to slow atherogenesis and the development of microvascular complications in diabetes, are obvious: Vitamin E-monotherapy, while blocking oxidative and AGE-modification of LDL, is unable to inhibit its AP-formation. As a consequence, tocopherol is susceptible to increased consumption by AP-associated radical production in hyperglycemic patients, which could be checked in part by the tocopherol-protecting agent troglitazone and/or by acetylsalicylic acid.     

Binding of long-term glycated low density lipoprotein and AGE-albumin by peripheral monocytes and endothelial cells

Modification of low density lipoprotein (LDL) and plasma or tissue proteins by non-enzymatic glycation culminating in the formation of advanced glycation endproducts (AGEs) is one of the essential pathomechanisms leading to diabetes-associated long-term complications. We compared binding of glycated, glycoxidated and oxidated LDL by peripheral monocytes in activated and quiescent form. Interaction via specific receptors was different for glycated as compared to (glyc)oxidated LDL-modifications. In addition, binding of glycated LDL to quiescent and activated human umbilical vein endothelial cells was studied. In patients with insulin-dependent diabetes mellitus (IDDM), AGE-binding was significantly increased as compared to healthy individuals. Specific and non-specific monocyte binding mechanisms were detected, and both were significantly increased in IDDM patients. Specific and non-specific binding strategies possibly act in concert to eliminate circulating AGEs, which are instrumental in the development and progress of microangiopathic and macroangiopathic complications of diabetes mellitus.     

Why is glycated LDL more sensitive to oxidation than native LDL? A comparative study

It is well established that oxidative modification of low-density lipoprotein (LDL) plays a causal role in human atherogenesis and the risk of atherosclerosis is increased in patients with diabetes mellitus. To examine the influence of different agents which may influence LDL-glycation and oxidation, experiments including glycation with glucose, glucose 6-phosphate, metal chelators (EDTA) and antioxidants (BHT) were performed. The influence of time dependence on the glycation process and the alteration of the electrophoretic mobility of LDL under diverse glycation and/or oxidation conditions was also investigated. The formation of conjugated dienes and levels of lipid peroxides in these different LDL-modifications were estimated. The copper-induced oxidation of LDL in vitro was determined by measurement of thiobarbituric acid reactive substances (TBARS) and expressed as nmol MDA/mg of LDL protein. We found that glycated LDL is more prone to oxidation than native LDL. Using native LDL, the maximal oxidation effect was found to reach a value of 49.72 nmol MDA/mg protein after 8 h. The maximum oxidation of the 31 days, glycated LDL with glucose was 71.76 nmol MDA/mg protein amounting to 144.33% of the value found for native LDL. In the case of glucose 6-phosphate glycation, the maximum oxidation under the same conditions amounted to 173.77% of the value found for native LDL. To measure the extent of glycation, fluorescence of advanced glycation end products (AGEs) was determined (370 nm excitation and 440 nm emission). The most potent glycation agent was glucose 6-phosphate leading to the formation of very high amounts of AGEs. This process was promoted in the absence of EDTA, which prevents the oxidative cleavage of modified Amadori products (ketoamines) to AGEs. We therefore conclude that both processes, glycation and oxidation, result in the modification of LDL. The lower the glycation-rate (+/- EDTA) as measured by relative fluorescence units RFU (generation of AGEs), the lower the additional oxidation rate after glycation as measured by TBARS (generation of MDA equivalents). Glycation and/or oxidation change the electrophoretic mobility of LDL.     

The Src/PLC/PKC/MEK/ERK signaling pathway is involved in aortic smooth muscle cell proliferation induced by glycated LDL

Low density lipoproteins (LDL) play important roles in the pathogenesis of atherosclerosis. Diabetes is associated with accelerated atherosclerosis leading to cardiovascular disease in diabetic patients. Although LDL stimulates the proliferation of arterial smooth muscle cells (SMC), the mechanisms are not fully understood. We examined the effects of native LDL and glycated LDL on the extracellular signal-regulated kinase (ERK) pathway. Addition of native and glycated LDL to rat aorta SMCs (RASMCs) stimulated ERK phosphorylation. ERK phosphorylation was not affected by exposure to the Ca2+ chelator BAPTA-AM but inhibition of protein kinase C (PKC) with GF109203X, inhibition of Src kinase with PP1 (5 microM) and inhibition of phospholipase C (PLC) with U73122/U73343 (5 microM) all reduced ERK phosphorylation in response to glycated LDL. In addition, pretreatment of the RASMCs with a cell-permeable mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059, 5 microM) markedly decreased ERK phosphorylation in response to native and glycated LDL. These findings indicate that ERK phosphorylation in response to glycated LDL involves the activation of PKC, PLC, and MEK, but is independent of intracellular Ca2+.     

Low density lipoprotein (LDL) inhibits histamine release from human mast cells

We examined the effect of low density lipoprotein (LDL) on histamine release from purified human lung mast cells. LDL inhibited anti-IgE- induced histamine release in a dose-dependent manner, with 100 micrograms/ml LDL-protein inhibiting histamine release by 53 +/- 8% (mean +/- SEM); half-maximal inhibition occurred at 40-80 micrograms/ml. LDL also inhibited calcium ionophore A23187-induced histamine release in a dose-dependent manner, with 1 mg/ml of LDL inhibiting histamine release by 83 +/- 9%; half maximal inhibition occurred at 220-280 micrograms/ml. Inhibition by LDL was time-dependent: half-maximal inhibition of anti-IgE- induced histamine release by LDL occurred at 30-50 minutes of incubation. The inhibitory effect of LDL was independent of buffer calcium concentrations (0-5 mM) or temperature (0-37 degrees C). These data are consistent with a newly defined immunoregulatory role for LDL.     

Glycated phosphatidylethanolamine promotes macrophage uptake of low density lipoprotein and accumulation of cholesteryl esters and triacylglycerols.

Non-enzymatic glycation of low density lipoprotein (LDL) has been suggested to be responsible for the increase in susceptibility to atherogenesis of diabetic individuals. Although the association of lipid glycation with this process has been investigated, the effect of specific lipid glycation products on LDL metabolism has not been addressed. This study reports that glucosylated phosphatidylethanolamine (Glc-PtdEtn), the major LDL lipid glycation product, promotes LDL uptake and cholesteryl ester (CE) and triacylglycerol (TG) accumulation by THP-1 macrophages. Incubation of THP-1 macrophages at a concentration of 100 micrograms/ml protein LDL specifically enriched (10 nmol/mg LDL protein) with synthetically prepared Glc-PtdEtn resulted in a significant increase in CE and TG accumulation when compared with LDL enriched in non-glucosylated PtdEtn. After a 24-h incubation with LDL containing Glc-PtdEtn, the macrophages contained 2-fold higher CE (10.11 +/- 1.54 micrograms/mg cell protein) and TG (285.32 +/- 4.38 micrograms/mg cell protein) compared with LDL specifically enriched in non-glucosylated PtdEtn (CE, 3.97 +/- 0.95, p < 0.01 and TG, 185.57 +/- 3.58 micrograms/mg cell protein, p < 0.01). The corresponding values obtained with LDL containing glycated protein and lipid were similar to those of LDL containing Glc-PtdEtn (CE, 11.9 +/- 1.35 and TG, 280.78 +/- 3.98 micrograms/mg cell protein). The accumulation of both neutral lipids was further significantly increased by incubating the macrophages with Glc-PtdEtn LDL exposed to copper oxidation. By utilizing the fluorescent probe, 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI), a 1.6-fold increase was seen in Glc-PtdEtn + LDL uptake when compared with control LDL. Competition studies revealed that acetylated LDL is not a good competitor for DiI Glc-PtdEtn LDL (5-6% inhibition), whereas glycated LDL gave an 80% inhibition, and LDL + Glc-PtdEtn gave 93% inhibition of uptake by macrophages. These results indicate that glucosylation of PtdEtn in LDL accounts for the entire effect of LDL glycation on macrophage uptake and CE and TG accumulation and, therefore, the increased atherogenic potential of LDL in hyperglycemia.     

In vitro oxidized and glycated human low-density lipoprotein particles characterized by capillary zone electrophoresis

A simple capillary zone electrophoresis (CZE) method was used to determine native, in vitro Cu(2+) and glucose modified low-density lipoprotein (LDL) particles for four healthy subjects. The LDL electropherograms are highly reproducible with good precisions of effective mobility and peak area. The native LDL capillary electrophoresis (CE) profile shows a major peak with lower mobility and two minor peaks with higher mobilities. For three-hour Cu(2+) oxidation, one major peak with mobility close to that of the native major peak, and one minor peak with mobility extending to -47 x 10(-5)cm(2)V(-1)s(-1) appear. For eighteen-hour Cu(2+) oxidation, one major peak with mobility much higher than that of the native major peak appears. As the reaction time for LDL and Cu(2+) increases from 0 to 24h, effective mobility of the LDL major peak increases, suggesting that LDL particles become more negatively charged and oxidized as the time increases. The in vitro glycated LDL particles are characterized by a major peak and two minor peaks. Mobility of the major peak is close to that of native major peak, but the second minor peak is much more negatively charged with mobility extending to -53 x 10(-5)cm(2)V(-1)s(-1). Native, oxidized and glycated LDL particles show distinctive differences in their CZE profiles. Agarose electrophoresis shows that the charge to mass ratios of native, three-hour Cu(2+) and glucose modified LDL particles are similar, but that of eighteen-hour Cu(2+) oxidized LDL particles is higher.