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Aims: Cardiovascular events remain the leading cause of death in Western world. Atherosclerosis is the most common underlying complication driven by low-density lipoproteins (LDL) disturbing vascular integrity. Carbamylation of lysine residues, occurring primarily in the presence of chronic kidney disease (CKD), may affect functional properties of lipoproteins; however, its effect on endothelial function is unknown., Methods and Results: Low-density lipoprotein from healthy donors was isolated and carbamylated. Vascular reactivity after treatment with native LDL (nLDL) or carbamylated LDL (cLDL) was examined in organ chambers for isometric tension recording using aortic rings of wild-type or lectin-like-oxidized LDL receptor-1 (LOX-1) transgenic mice. Reactive oxygen species (ROS) and nitric oxide (NO) production were determined using electron spin resonance spectroscopy. The effect of LDL-carbamyl-lysine levels on cardiovascular outcomes was determined in patients with CKD during a median follow-up of 4.7 years. Carbamylated LDL impaired endothelium-dependent relaxation to acetylcholine or calcium-ionophore A23187, but not endothelium-independent relaxation to sodium nitroprusside. In contrast, nLDL had no effect. Carbamylated LDL enhanced aortic ROS production by activating NADPH-oxidase. Carbamylated LDL stimulated endothelial NO synthase (eNOS) uncoupling at least partially by promoting S-glutathionylation of eNOS. Carbamylated LDL-induced endothelial dysfunction was enhanced in LOX-1 transgenic mice. In patients with CKD, LDL-carbamyl-lysine levels were significant predictors for cardiovascular events and all-cause mortality., Conclusions: Carbamylation of LDL induces endothelial dysfunction via LOX-1 activation and increased ROS production leading to eNOS uncoupling. This indicates a novel mechanism in the pathogenesis of atherosclerotic disease which may be pathogenic and prognostic in patients with CKD and high plasma levels of cLDL., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

It has long been considered that oxidized low-density lipoprotein (oxLDL) causes endothelial dysfunction and is remarkably related to the development of atherosclerosis. However, the effect of oxLDL at very low concentration (<10μg/ml) on the endothelial cells remains speculative. Nitric oxide (NO) has a crucial role in the endothelial cell function. In this study, we investigated the effect of oxLDL at low concentration on NO production and proliferation, migration, tube formation of the human coronary artery endothelial cells (HCAEC). Results showed that oxLDL at 5μg/ml enhanced HCAEC proliferation, migration and tube formation. These phenomena were accompanied by an increased intracellular NO production. l-NAME (a NOS inhibitor), LY294002 and wortmannin (PI3K inhibitors) could abolish oxLDL-induced angiogenic effects and prevent NO production in the HCAEC. The phosphorylation of Akt, PI3K and eNOS were up-regulated by oxLDL, which was attenuated by LY294002. Our results suggested that oxLDL at low concentration could promote in-vitro angiogenesis and activate nitric oxide synthesis through PI3K/Akt/eNOS pathway in HCAEC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rationale: Atherosclerosis proceeds through a multistep reaction that begins with endothelial injury caused by a host of stress signals, among which oxidized low-density lipoprotein (oxLDL) plays a critical role. OxLDL disrupts normal functionality of the endothelium by upregulating adhesion molecules (eg, ICAM-1) and concomitantly downregulating endothelial nitric oxide synthase (eNOS) expression. The transcriptional modulator that mediates the cellular response to oxLDL remains largely obscure., Objective: Our goal was to determine whether myocardin-related transcription factor (MRTF)-A, a key protein involved in the transcriptional regulation of smooth muscle cell phenotype, is responsible for the endothelial injury by oxLDL, and, if so, how MRTF-A promotes the proatherogenic agenda initiated by oxLDL., Methods and Results: OxLDL stimulated the expression of MRTF-A in endothelial cells as evidenced by Western blotting and immunofluorescence. Overexpression of MRTF-A synergistically enhanced the induction of ICAM-1 and suppression of eNOS by oxLDL. In contrast, disruption of MRTF-A, either by small interfering RNA or dominant negative mutation, abrogated the pathogenic program triggered by oxLDL. Finally, chromatin immunoprecipitation assays indicate that oxLDL preferentially augmented MRTF-A binding to ICAM-1 and eNOS promoters and that MRTF-A drove differential epigenetic alterations taking place on these promoters in response to oxLDL., Conclusions: Therefore, our data provide the first demonstration that MRTF-A is critically linked to pivotal pathophysiological events in the vascular endothelium.

Oxidized low-density lipoprotein (oxLDL) is a major factor in development of atherosclerosis. Our earlier studies have shown that exposure of endothelial cells (EC) to oxLDL increases EC stiffness, facilitates the ability of the cells to generate force, and facilitates EC network formation in three-dimensional collagen gels. In this study, we show that oxLDL induces a decrease in lipid order of membrane domains and that this effect is inversely correlated with endothelial stiffness, contractility, and network formation. Local lipid packing of cell membrane domains was assessed by Laurdan two-photon imaging, endothelial stiffness was assessed by measuring cellular elastic modulus using atomic force microscopy, cell contractility was estimated by measuring the ability of the cells to contract collagen gels, and EC angiogenic potential was estimated by visualizing endothelial networks within the same gels. The impact of oxLDL on endothelial biomechanics and network formation is fully reversed by supplying the cells with a surplus of cholesterol. Furthermore, exposing the cells to 7-keto-cholesterol, a major oxysterol component of oxLDL, or to another cholesterol analog, androstenol, also results in disruption of lipid order of membrane domains and an increase in cell stiffness. On the basis of these observations, we suggest that disruption of lipid packing of cholesterol-rich membrane domains plays a key role in oxLDL-induced changes in endothelial biomechanics.

The endothelium mediates relaxations (dilatations) of the underlying vascular smooth muscle cells. The endothelium-dependent relaxations are due to the release of non-prostanoid vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also release substances (endothelium-derived hyperpolarizing factor, EDHF) that cause hyperpolarization of the cell membrane of the underlying vascular smooth muscle. The release of EDRF from the endothelium can be mediated by both pertussis toxin-sensitive G(i) (alpha(2)-adrenergic activation, serotonin, thrombin) and insensitive G(q) (adenosine diphosphate, bradykinin) coupling proteins. The ability of the endothelial cell to release relaxing factors can be upregulated by impregnation with estrogens, exercise and antioxidants, and down-regulated by oxidative stress and increased presence of oxidized LDL. Following injury or apoptotic death, the endothelium regenerates. However, in regenerated endothelial cells, there is an early selective loss of the pertussis-toxin sensitive mechanisms of EDRF-release. Functional studies suggest that abnormal handling of LDL because of increased oxidative stress play a key role in this selective loss. Genomic analysis demonstrates the emergence of fatty acid binding protein-A (A-FBP) and metalloproteinase-7 (MMP7) in regenerated endothelial cells. The reduced release of NO resulting from the endothelial dysfunction in regenerated areas creates a locus minoris resistentiae which favors the occurrence of vasospasm and thrombosis as well as the initiation of atherosclerosis.

Platelets are involved in the initiation of atherosclerosis by adherence to inflamed endothelium. Monocytes bind to these platelets and transmigrate into the vessel wall, transforming into macrophages and foam cells. We have previously shown that lipid-laden platelets are phagocytosed by macrophages. In this study we investigated the functional consequences of oxidized low-density lipoprotein (oxLDL) uptake on platelet function and interaction with the endothelium. Human platelets were isolated from healthy donors and activated by adenosine diphosphate. Immunofluorescence microscopy and flow cytometry revealed that oxLDL is located intracellularly in vesicles. With mepacrine costaining and confocal microtomography, we were able to identify dense granules as the vesicles that contain oxLDL. OxLDL-laden platelets induced intercellular adhesion molecule 1 expression in endothelial cells more than exogenous native LDL, oxLDL, and oxLDL-negative platelets. Furthermore, oxLDL-laden platelets induced foam cell development from CD34(+) progenitor cells. On endothelial regeneration, oxLDL-laden platelets had the opposite effect: The number of CD34(+) progenitor cells (colony-forming units) able to transform into endothelial cells was significantly reduced in the presence of oxLDL-platelets, whereas native LDL had no effect. Our results demonstrate that activated platelets internalize oxLDL and that oxLDL-laden platelets activate endothelium, inhibit endothelial regeneration, and promote foam cell development. Platelet oxLDL contributes significantly to vascular inflammation and is able to promote atherosclerosis., (Thieme Medical Publishers.)

Background: Endothelial cell hyperpermeability is a proposed mechanism of increased lipid insudation into the vessel walls of allografts. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability and its expression is upregulated in human heart allografts. The goal of these experiments was to investigate the effects of VEGF on low-density lipoprotein (LDL) permeability through confluent monolayers of human cardiac microvascular endothelial cells (HCMEC) in vitro., Methods: VEGF mRNA and protein expression was characterized in coronary arteries from cardiac allograft vasculopathy patients as compared with healthy controls using in situ hybridization and immunohistochemical staining of sub-adjacent sections. HCMEC were grown to confluence and treated with VEGF-A(121) or VEGF-A(165). Permeability of LDL in confluent endothelial monolayers was measured using fluorometry. Transendothelial electrical resistance (TER) measurements were used to indirectly measure the tight junctional status. Immunocytochemical staining was performed to visualize changes in CD31 and zonula occludens-1., Results: We observed significant increases in VEGF expression within the superficial and deep intima and media of coronaries from allografts, as compared with controls. In vitro treatment with VEGF-A(121) and VEGF-A(165) significantly increased LDL passage through endothelial monolayers. We further showed that VEGF-A(121) and VEGF-A(165) caused significant decreases in TER at 2 to 4 hours post-treatment. Also, VEGF induced disruption of tight junctions, resulting in an increase in the intercellular gap formation., Conclusions: These results demonstrate that VEGF increases low-density lipoprotein permeability through endothelial monolayers, and this effect is correlated with VEGF-induced disruption of endothelial tight junctions resulting in the formation of intercellular gaps.

In response to physiologic stimuli, endothelial cells dynamically regulate arterial vascular tone by producing vasodilators and vasoconstrictors. Risk factors for atherosclerosis, such as diabetes, smoking, hypercholesterolemia, and hypertension, interfere with this response, promoting endothelial dysfunction and atherosclerosis. This review explores whether oxidative stress might be a common feature of both endothelial dysfunction and atherosclerosis. Using biomarkers to assess endothelial function might provide insights into the pathways for oxidative stress in vascular disease. However, currently available markers of oxidative stress and endothelial function are unsuitable for routine clinical use because they are too expensive and inadequately validated. Thus, there is a need to develop and validate new markers that could be used to both measure oxidative stress and monitor therapies that specifically interrupt oxidative pathways in vascular tissue. Such markers might eventually help to identify susceptible individuals at a stage when cardiovascular complications could be prevented.

Oxidized LDL (OxLDL) is a proatherogenic lipoprotein, accumulating in the vascular wall and contributing to the pathogenesis of vascular dysfunction early in the development of atherosclerosis. Enhanced serum levels of OxLDL, as well as antibodies against its epitopes, are predictive for endothelial dysfunction and coronary heart disease. While enhanced oxidative stress is one factor triggering formation of OxLDL, OxLDL itself has been identified as a potent stimulus for vascular oxygen radical formation, causing a vicious circle. OxLDL-induced O(2)(-) formation, largely through activation of NADPH oxidase, but also through uncoupling of endothelial NO-synthase and through direct O(2)(-) release, leads to endothelial dysfunction. Furthermore, OxLDL-induced O(2)(-) formation has a strong impact on tissue remodeling, resulting in either cell growth - proliferation or hyperplasia - or apoptotic cell death. The effect of OxLDL on cell cycle regulation is mediated by activation of the small GTPase RhoA and consequent regulation of p27(KIP1), a key enzyme of the cell cycle. In addition, OxLDL-induced activation of RhoA sensitizes the contractile apparatus of the vessel wall, enhancing the contractile tonus and favoring vasospasm. Thus, through a variety of mechanisms, OxLDL importantly contributes to vascular dysfunction and remodeling.

Aim: To explore the associations of LDL (low-density lipoprotein) particle size and oxidized LDL with endothelium-dependent function of the forearm microcirculation in diabetes., Methods: Endothelium-dependent function was examined in 43 middle-aged men and women with type 2 diabetes and 10 age-matched controls. All received aspirin to inhibit endothelial cyclo-oxygenase. Forearm blood flow (FBF) was measured using venous occlusion plethysmography with separate administration of acetylcholine (ACh) and bradykinin (BK) into the brachial artery. Endothelium-independent function was assessed using sodium nitroprusside (SNP). N(G)-monomethyl-L-arginine (L-NMMA) was co-infused with ACh (ACh + L-NMMA) and BK (BK + L-NMMA) to assess non-NO-mediated contributions to endothelium-dependent function., Results: Subjects with diabetes had impaired endothelium-dependent and endothelium-independent function compared with controls (p < 0.01 for ACh, BK and SNP). In multivariate regression analysis, LDL size (r = 0.41 and p = 0.007), oxidized LDL (r = -0.41 and p = 0.007) and duration of diabetes (r = -0.37 and p = 0.02) predicted FBF response to ACh independently of age, gender and systolic blood pressure. There were no associations between LDL size, oxidized LDL, duration of diabetes and FBF response to BK, SNP, ACh + L-NMMA or BK + L-NMMA., Conclusion: In type 2 diabetes, small dense LDL particles, duration of diabetes and oxidized LDL may independently contribute to endothelial dysfunction of the microcirculation. These disturbances may occur via a selective defect, because ACh and BK activate endothelial NO synthase via different G-protein signal transduction pathways.

A model system is presented using human umbilical vein endothelial cells (HUVECs) to investigate the role of homocysteine (Hcy) in atherosclerosis. HUVECs are shown to export Hcy at a rate determined by the flux through the methionine/Hcy pathway. Additional methionine increases intracellular methionine, decreases intracellular folate, and increases Hcy export, whereas additional folate inhibits export. An inverse relationship exists between intracellular folate and Hcy export. Hcy export may be regulated by intracellular S-adenosyl methionine rather than by Hcy. Human LDLs exposed to HUVECs exporting Hcy undergo time-related lipid oxidation, a process inhibited by the thiol trap dithionitrobenzoate. This is likely to be related to the generation of hydroxyl radicals, which we show are associated with Hcy export. Although Hcy is the major oxidant, cysteine also contributes, as shown by the effect of glutamate. Finally, the LDL oxidized in this system showed a time-dependent increase in uptake by human macrophages, implying an upregulation of the scavenger receptor. These results suggest that continuous export of Hcy from endothelial cells contributes to the generation of extracellular hydroxyl radicals, with associated oxidative modification of LDL and incorporation into macrophages, a key step in atherosclerosis. Factors that regulate intracellular Hcy metabolism modulate these effects.

It is now widely accepted that oxidant stress and the ensuing endothelial dysfunction play a key role in the pathogenesis of atherosclerosis and cardiovascular diseases. The mitochondrial respiratory chain is the major source of reactive oxygen species as byproducts of normal cell respiration. Mitochondria may also be important targets for reactive oxygen species, which may damage mitochondrial lipids, enzymes and DNA with following mitochondrial dysfunction. Free cholesterol, oxidized low-density lipoprotein and glycated high-density lipoprotein are further possible causes of mitochondrial dysfunction and/or apoptosis. Moreover, in patients with mitochondrial diseases, vascular complications are commonly observed at an early age, often in the absence of traditional risk factors for atherosclerosis. We propose that mitochondrial dysfunction, besides endothelial dysfunction, represents an important early step in the chain of events leading to atherosclerotic disease., (Copyright 2005 S. Karger AG, Basel.)

Objective: To investigate the effects of low concentrations of oxidized LDL (oxLDL) on the proliferation and spontaneous motility of human coronary artery endothelial cells (HCAEC) in culture., Methods: Cultures of HCAEC were treated with low concentrations of native LDL (nLDL) isolated from human plasma and with LDL minimally oxidized through different chemical methods; the effects were compared., Results: Native LDL had no deleterious effects on in vitro proliferation and motility of HCAEC; however, at its highest concentration and for a longer exposure, nLDL inhibited cell proliferation. The LDL chemically oxidized by spermine nonoate (SNO) and 3-morpholinylsydnonimine (SYN-1) had significant inhibiting effects on in vitro proliferation and motility of HCAEC, which were proportional to the greatest concentrations and degrees of oxidation of LDL., Conclusion: OxLDL has a cytotoxic effect, inhibiting the proliferation and spontaneous motility of HCAEC in culture. This effect is proportional to the concentration and degree of oxidation of LDL; native LDL is relatively innocuous.

Background: Impaired arterial dilatation response to nitroglycerin has been observed in adults with risk factors for atherosclerosis and in patients with established atherosclerotic disease. This defect parallels changes in vascular endothelial function and may be attributed to increased oxidative stress. Because atherosclerosis begins in childhood, we examined the correlates of nitrate-mediated dilatation (NMD) in children, including brachial artery endothelial function, oxidized LDL, and carotid artery intima-media thickness (IMT)., Methods and Results: Brachial artery flow-mediated endothelium-dependent dilatation (FMD) and nitrate-mediated smooth muscle function, IMT of the carotid bulb, and brachial artery and oxidized LDL were measured in 142 children (mean age, 11 years; range, 8 to 17 years), including 87 healthy children, 41 diabetic children, and 14 children with familial hypercholesterolemia. NMD correlated directly with FMD (r=0.46, P<0.001) and inversely with brachial artery baseline diameter (r=-0.36, P<0.001), age (r=-0.25, P=0.003), body mass index (r=-0.31, P<0.001), diabetes (r=-0.22, P=0.008), oxidized LDL (r=-0.18, P=0.045), and IMT (r=-0.33, P<0.001). In a multivariate regression model, the significant correlates for NMD were FMD response (beta=0.003, P<0.001), brachial artery diameter (beta=-0.03, P=0.01), oxidized LDL (beta=-0.07, P=0.02), and IMT (beta=-0.15, P=0.03)., Conclusions: Reduced endothelial function, increased oxidative stress, and preclinical carotid atherosclerosis are independent determinants of impaired NMD in children. These data thus suggest that primary nitrate tolerance occurs in children at risk for atherosclerosis.

Background: Oxidation of low density lipoprotein (LDL) has been implicated in infiltration of foam cells derived from circulating monocytes. Monocyte adhesion to endothelial cells and migration into dermis are essential steps for infiltration of foam cells., Objective: We investigated the role of adhesion molecules contributing to the process of monocyte adhesion to human dermal microvascular endothelial cells (HDMEC). Special attention was paid to the signal transduction for adhesion molecule expression induced by two distinct types of oxidized LDL., Methods: HDMEC were incubated with xanthoma tissue-modified LDL (x-LDL), a model of extravasated LDL oxidized in xanthoma lesions, or Cu(2+)-treated LDL (Cu-LDL), a model of oxidized LDL. Adhesion of U937 cells, a human monocytic leukemia cell line, to HDMEC and expression of endothelial cell adhesion molecules on HDMEC were examined. Signal transduction pathways for the adhesion molecule expression were evaluated by employing specific inhibitors., Results: x-LDL induced adhesion of U937 cells to HDMEC through vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by activating tyrosine kinase pathway. Cu-LDL up-regulated the adhesion through not only VCAM-1 and E-selectin but also intercellular cell adhesion molecule-1 (ICAM-1) by activating G(i) protein pathway., Conclusion: Extravasated and oxidized LDL in xanthoma lesions contributes to foam cell recruitment by activating tyrosine kinase pathway and inducing adhesion of monocytes to HDMEC through VCAM-1 and E-selectin. Cu-LDL, on the other hand, activates G(i) protein pathway and induces the adhesion through ICAM-1, VCAM-1 and E-selectin.

Under normal conditions, vascular endothelial cells are resistant to Fas-mediated apoptosis, although they express detectable Fas on their cell surface. Because oxidized Low density lipoprotein (Ox-LDL) is thought to promote atherogenesis, the potential role that Ox-LDL may play in Fas-mediated apoptosis was investigated in human umbilical vascular endothelial cells (HUVECs), focusing particularly on the involvement of the lectin-like Ox-LDL receptor-1 (LOX-1). HUVECs were treated with agonistic anti-Fas antibody (CH11) and Ox-LDL and then the degree of apoptosis was determined by cell death ELISA. Ox-LDL concentration-dependently sensitized Fas-mediated apoptosis. Flow cytometry demonstrated that Ox-LDL dose-dependently up-regulated cell surface Fas expression. On the other hand, treating HUVECs with Ox-LDL did not lead to any significant change in the expression of death mediators, including Fas, Fas ligand (FasL), FADD, and FLICE as assessed by multiplex polymerase chain reaction amplification. More importantly, these effects of Ox-LDL on Fas-mediated apoptosis were significantly blocked by a neutralizing LOX-1 monoclonal antibody, which can block LOX-1-mediated cellular uptake of Ox-LDL. Ox-LDL may be an important factor involved in the regulation of Fas-induced apoptosis via Ox-LDL/LOX-1 interaction in vascular endothelial cells. The results may provide insights into the pathogenesis of accelerated atherosclerosis in patients with hyperlipidemia.

Objectives: We examined the hypothesis that low-density lipoprotein (LDL) that is both oxidized and glycosylated potently downregulates the expression of endothelial nitric oxide synthase III (NOSIII) in human coronary endothelial cells., Background: Diabetes mellitus is accompanied by both oxidation and glycosylation of LDL, but the potential interaction of these processes or the pathophysiologic effects of these modified lipoproteins on arteries are poorly understood., Methods: Low-density lipoprotein was glycoxidized in vitro, and Western and Northern blot analyses were used to investigate NOSIII expression in human coronary endothelial cells. Nitric oxide (NO) bioactivity was represented by both basal and bradykinin-stimulated cellular cyclic guanosine monophosphate accumulation and L-citrulline conversion from L-arginine. Nuclear run-on experiments were performed to study the transcription rate of nascent NOSIII messenger ribonucleic acid (mRNA)., Results: Data showed a significant decrease in NOSIII expression after 24-h treatment with glycosylated low-density lipoprotein (glycLDL) and oxidized low-density lipoprotein (oxLDL). Accordingly, we observed a significant dose-dependent reduction in NO bioactivity (p < 0.05 to p < 0.001 vs. untreated cells, native low density lipoprotein [nLDL], glycLDL, and oxLDL). Glyc-oxLDL did not reduce the half-life of NOSIII mRNA or significantly enhance L-citrulline conversion. Nuclear run-on experiments showed that high doses of glyc-oxLDL can reduce the transcription rate of nascent NOSIII mRNA (densitometric analysis revealed a reduction of 25% [p < 0.05 vs. untreated cells, nLDL, and glycLDL] after treatment of cells with 300 microg/ml glyc-oxLDL). The effects of glyc-oxLDL are not related to the higher levels of oxidative compounds in comparison to those of oxLDL., Conclusions: These results indicate that glyc-oxLDL, per se, may influence signal transduction pathways involving NO-mediated regulatory signals and NOSIII activity in human endothelial cells. This phenomenon can adversely influence the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in diabetic patients.

Background and Aim: Evidence for apoptosis has been found in advanced atherosclerotic lesions, but the factors triggering it are poorly understood. Oxidised low-density lipoproteins (LDLs) are cytotoxic to a variety of cells and induce the apoptosis of smooth muscle cells (SMC), fibroblast, macrophages and endothelial cells in vitro. The aim of this study was to investigate apoptotic cell death in the early phases of aortic atherosclerosis in rabbits, and whether oxidised LDLs colocalize ex vivo with apoptotic cells in atherosclerotic lesions in cholesterol-fed rabbits., Methods and Results: Male albino New Zealand rabbits were fed a standard diet or a diet containing 1.2% cholesterol for 60 days. The aortic arch of each animal was sectioned and stained with antibodies against SMC, endothelial cells, macrophages and oxidised LDLs or for proteins involved in apoptotic pathways such as Fas, Bax, Bcl2, and caspase 3. The nuclei in adjacent sections were stained with Hoechst 33258, TUNEL and for the proliferating cell nuclear antigen (PCNA). Early atherosclerotic lesions were characterised by intimal thickening and the presence of SMC and macrophages. The percentage of apoptotic cells, calculated as the ratio of TUNEL-positive nuclei to total nuclei was 32.6 +/- 3.73% in the lesions and 55.9 +/- 2.36% in the endothelium. As it has been reported that nuclei undergoing active gene transcription can be TUNEL positive, we evaluated the percentage of PCNA-positive cells, which proved to be 45.2 +/- 4.68% along the endothelium and 22.3 +/- 2.7% in the intima. The true percentage of apoptotic cells was therefore about 10% in both cases. Fas, Bax and caspase3 signals were mainly located in the endothelium and SMC proximal to the lumen, whereas Bcl2 colocalized with macrophages and SMC deeper in the lesions. Abundant oxidised LDL epitopes were detected in areas of lipid accumulation and along the endothelium, mainly in the areas in which TUNEL and PCNA-positive cells were localised., Conclusions: Our findings may be taken as ex vivo indications of an apoptotic and proliferating role of oxidised LDLs as previously shown in vitro, and may at least partially account for the endothelial dysfunction that can be rapidly induced by hypercholesterolemia.

Objective: Hypercholesterolemia induces endothelial dysfunction, a hallmark of the atherosclerotic process, modulating the expression of key genes in vascular endothelial cells., Methods and Results: By differential display analysis, we have studied the effect of high concentrations of native low density lipoprotein (LDL) on endothelial gene expression. mRNA levels of lysyl oxidase (LOX), an enzyme involved in collagen and elastin cross-linking, were downregulated by LDL treatment in endothelial cells in a dose- and time-dependent manner (80% of inhibition by 180 mg/dL LDL for 24 hours). This reduction of LOX expression was associated with a decrease in LOX activity (40% and 54% of inhibition after 24 and 48 hours of LDL treatment, respectively). LOX mRNA half-life was not modified by LDL, but transcriptional inhibition blocked the effect of LDL. Inhibition of LOX activity by either LDL or beta-aminopropionitrile, an inhibitor of LOX, increased endothelial permeability (192+/-0.19- and 3.37+/-0.74-fold, respectively). Interestingly, a reduction in LOX expression (3.5-fold) was observed in vivo in the vascular wall of hypercholesterolemic pigs., Conclusions: These findings suggest that LDL downregulation of LOX could contribute to the endothelial dysfunction caused by hypercholesterolemia, thus contributing to atherosclerotic plaque formation.

Numerous pathologies may involve toxic side effects of free heme and heme-derived iron. Deficiency of the heme-catabolizing enzyme, heme oxygenase-1 (HO-1), in both a human patient and transgenic knockout mice leads to an abundance of circulating heme and damage to vascular endothelium. Although heme can be directly cytotoxic, the present investigations examine the possibility that hemoglobin-derived heme and iron might be indirectly toxic through the generation of oxidized forms of low-density lipoprotein (LDL). In support, hemoglobin in plasma, when oxidized to methemoglobin by oxidants such as leukocyte-derived reactive oxygen, causes oxidative modification of LDL. Heme, released from methemoglobin, catalyzes the oxidation of LDL, which in turn induces endothelial cytolysis primarily caused by lipid hydroperoxides. Exposure of endothelium to sublethal concentrations of this oxidized LDL leads to induction of both HO-1 and ferritin. Similar endothelial cytotoxicity was caused by LDL isolated from plasma of an HO-1-deficient child. Spectral analysis of the child's plasma revealed a substantial oxidation of plasma hemoglobin to methemoglobin. Iron accumulated in the HO-1-deficient child's LDL and several independent assays revealed oxidative modification of the LDL. We conclude that hemoglobin, when oxidized in plasma, can be indirectly cytotoxic through the generation of oxidized LDL by released heme and that, in response, the intracellular defense-HO-1 and ferritin-is induced. These results may be relevant to a variety of disorders-such as renal failure associated with intravascular hemolysis, hemorrhagic injury to the central nervous system, and, perhaps, atherogenesis-in which hemoglobin-derived heme may promote the formation of fatty acid hydroperoxides.

Calcium channel blockade has been shown to inhibit experimental atherosclerosis, and early clinical trials suggest that it also reduces atherosclerosis in humans. However, the mechanisms underlying the direct protective effect of calcium channel blockade on endothelial cell injury are not fully understood. The apoptosis of endothelial cells induced by oxidized low-density lipoproteins (oxLDL) may provide a mechanistic clue to the "response-to-injury" hypothesis of atherogenesis. Here we report that the calcium channel blocker, nifedipine, prevents the apoptosis of human umbilical venous endothelial cells (HUVECs) induced by oxLDL via downregulation of the endothelial receptor for oxidized LDL (LOX-1) and inhibition of CPP32-like protease activity. The incubation of HUVEC with oxLDL increased LOX-1 mRNA levels and CPP32-like protease activity, and induced apoptosis. Preincubation of HUVEC with nifedipine before incubation with oxLDL significantly suppressed the increase in LOX-1 mRNA levels and CPP32-like protease activity, preventing apoptosis in a dose-dependent manner. These results suggest that nifedipine blocks the suicide pathway leading to the apoptosis of endothelial cells by decreasing LOX-1 mRNA levels and CPP32-like protease activity. Thus, nifedipine seems to play a protective role against the "response-to-injury" hypothesis of atherogenesis.

Wistar rats were fed Se-deficient (0.038 mg/kg diet) and adequate (0.326 mg/kg diet) diets for 13 weeks. The blood Se content, blood and vascular wall glutathione peroxidase (GPx) activity, serum high-density lipoprotein cholesterol (HDL-C) level and plasma prostacyclin (PGI(2)) concentration were decreased significantly, and the blood lipid peroxide (LPO) concentration, serum low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC) level and plasma thromboxane A(2) (TXA(2)), content were increased significantly in Se-deficient rats compared with Se-adequate group, respectively. Furthermore the Se-deficient and adequate rats were given 5 mg/kg of cholestane-3 beta, 5 alpha, 6 beta -triol (3-triol) or vehicle only. Twenty four hours after treatment, the plasma PGI(2) level was decreased in Se-adequate rats infused 3-triol (+3 triol), meanwhile, the level in Se- deficient +3-triol group was much lower than that in Se-adequate +3-triol group. Compared with Se-adequate group, plasma TXA(2) content in Se-adequate +3-triol group had no significantly difference, but in Se- deficient rats infused 3-triol, plasma TXA(2) content was much higher than that in Se-adequate +3-triol group. The plasma ET concentration in Se-deficient group decreased slightly, but the concentration in Se-adequate +3-triol group increased significantly with respect to Se-deficient group. Although plasma ET concentration in Se-deficient group +3-triol did not increase, it was significantly lower than that in Se-adequate +3-triol group. The luminal surfaces of aorta thoracica of experimental rats were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Observed by SEM, the luminal surface of aorta of Se-deficient rats showed few crater-like defects due to the disruption of endothelial cell. Se-adequate +3-triol group showed some crater-like defects on the their aorta luminal surface, but the luminal surface of Se-deficient +3-triol group exhibited numerous crater-like defects and appeared sponge-like as well as platelets adhering followed by thrombus formation in focal area of extensive endothelial damage. TEM studies also showed that the endothelium of aorta of Se deficient +3-triol group had more frequent lesion where endothelial cell plasma were swelling with profuse intracellular edema and some vacuoles were seen in cytoplasm. In severely injured areas, endothelial integrity was completely destroyed and smooth muscle cells were proliferating and migrated to the endomembrane. Thus, we can conclude that Se or selenoproteins in the vascular wall plays an important role in cytoprotection against cholesterol oxide-induced vascular damage in rats.

Exposure of endothelial cells to oxidized low-density lipoprotein (oxLDL) leads to diverse cellular effects, including induction of the intracellular antioxidant GSH. It is not known whether lipid-or protein-derived oxidation products cause GSH induction and whether this involves increased activity of the key enzyme in its synthesis, glutamate-cysteine ligase (GCL). Furthermore, the effect of oxLDL exposure on the cell's ability to combat oxidative stress has not been previously examined. In the present study we found that, in bovine aortic endothelial cells, LDL or 1-palmitoyl-2-arachidonyl phosphatidylcholine oxidized by different reactive oxygen and nitrogen species induced GSH synthesis. However, prevention of GSH synthesis during exposure to oxLDL caused extensive cell death. The mediator causing GSH induction was shown to be a polar lipid and resulted in the increased activity of GCL as well as increased protein levels of the regulatory subunit of GCL. Pretreatment with both oxLDL and the polar lipid subfraction of the oxLDL protected cells against the toxicity of 2,3-dimethoxynaphthoquinone (DMNQ), a superoxide- and H(2)O(2)-forming compound. The potential of a low level of lipid peroxidation products to initiate cytoprotective pathways are discussed.

To find out whether concentration polarization of low-density lipoprotein (LDL) occurs at the surface of a vascular endothelium or not, transport of LDL in flowing blood to an water-permeable endothelium was studied theoretically by means of CFD. Calculations were carried out for an endothelium exposed to a Couette flow by assuming that the surface geometry of the endothelium could be expressed by a cosine function. Two typical cases were considered for the permeability of endothelium to water; one was uniform permeability everywhere in the endothelium, and the other was uneven permeability which was augmented at the intercellular junction. It was found that, in both cases, the surface concentration of LDL increased in going distally from the entrance, taking locally high and low values at the valleys and hills of the endothelium, respectively, and the variation was larger in the case of endothelium with uneven permeability. These results clearly showed that concentration polarization of LDL which might affect the uptake of LDL by the arterial wall certainly occurs at the surface of the endothelium even if the flow is disturbed microscopically by the uneven surface of the endothelium.

The imbalance in pro- and anti-oxidant activity causes endothelial dysfunction, which precedes and exacerbates atherosclerosis. LDL-apheresis improves ischemic symptom in patients with hypercholesterolemia and regresses atherosclerosis. Whereas, patients who undergo hemodialysis experience accelerated atherosclerosis and premature death even though their cholesterol level is known to be normal. We investigated the association between oxidative modification and endothelial function during a single session of blood purification therapies, LDL-apheresis and hemodialysis. To examine the effect of a single session of LDL-apheresis on endothelial function in patients with hypercholesterolemia, we measured forearm blood flow (FBF) by strain gauge plethysmography before and after LDL-apheresis, while infusing acetylcholine (ACh) and sodium nitroprusside (SNP). The single session of LDL-apheresis reduced oxidized-LDL. Although ACh and SNP increased FBF dose-dependently before and after LDL-apheresis, ACh response was significantly augmented without changes in responses to SNP. The plasma level of oxidized LDL correlated with the degree of ACh-induced vasodilatation. To examine the effect of a single session of hemodialysis on endothelial function in patients with end stage renal disease, we estimated flow-mediated vasodilation (FMD) during reactive hyperemia using a high-resolution ultrasonography, before and after a single session of hemodialysis. We also investigated the effect of anti-oxidative modification during hemodialysis using an a-tocopherol acetate-coated or a non-coated cellulose dialyzer. Non-specific endothelium independent vasodilation was measured after sublingual glyceryltrinitrate spray (GTN; 0.3 mg) administration in each session. A single session of hemodialysis increased plasma level of oxidized LDL. Although hemodialysis treatment did not affect GTN-induced vasodilation, a single session of hemodialysis impaired the endothelium-dependent vasodilation. Treatment with vitamin E-coated-dialyzer restored endothelium-dependent vasodilation, which was accompanied by decrease in oxidized LDL. Thus, endothelial function was impaired by acute increase in oxidative stress and was restored by anti-oxidant therapy. Our results suggest the importance of controlling oxidative stress in addition to classical risk factors to preserve human endothelial function.

Background: Oxidized LDL (oxLDL) inhibits angiogenesis in part by downregulating endothelial basic fibroblast growth factor (bFGF). To determine the mechanism of the downregulation, we investigated the signal transduction pathway involving potential phospholipid mediators., Methods and Results: Cultured bovine aortic endothelial cells were incubated with PBS (lipoprotein-free control), LDL, or copper oxLDL under serum-free conditions. At 24 hours, oxLDL (50 microg/mL) decreased bFGF mRNA (Northern blot), bFGF protein (Western blot and ELISA), and concomitant DNA synthesis, all by 40% to 50% compared with PBS. LDL had no effect. Pretreating the cells with 100 ng/mL pertussis toxin (PTX) for 18 hours before oxLDL exposure almost completely blocked the inhibitory effects of oxLDL. In contrast, inhibiting other major cellular signal transduction pathways with PD-98059 (mitogen-activated protein kinase kinase inhibitor), HA-1004 (inhibitor of cGMP- and cAMP-dependent protein kinase), or Ro-31-8220 (protein kinase C inhibitor) or chelating intracellular Ca(2+) with BAPTA-AM failed to attenuate any of the oxLDL effects assayed. Addition to the cultures of WEB 2086, a specific antagonist of the PTX-sensitive G protein-coupled platelet-activating factor (PAF) receptor, blocked the action of oxLDL. Whereas PAF dispersed in the culture medium failed to produce oxLDL-like effects, degradation of PAF and PAF-like phospholipids accumulated in oxLDL with a recombinant human PAF acetylhydrolase eliminated the inhibitory effects of oxLDL on bFGF expression and DNA synthesis., Conclusions: OxLDL suppresses endothelial bFGF expression and DNA synthesis through a PTX-sensitive heterotrimeric G-protein pathway involving mediator phospholipids similar, but not identical, to PAF.

Vascular endothelial growth factor (VEGF) has been recognized as an angiogenic factor that induces endothelial proliferation and vascular permeability. Recent studies have also suggested that VEGF can promote macrophage migration, which is critical for atherosclerosis. We have reported that VEGF is remarkably expressed in activated macrophages, endothelial cells, and smooth muscle cells within human coronary atherosclerotic lesions, and we have proposed the significance of VEGF in the progression of atherosclerosis. To clarify the mechanism of VEGF expression in atherosclerotic lesions, we examined the regulation of VEGF expression by oxidized low density lipoprotein (Ox-LDL), which is abundant in atherosclerotic arterial walls. A recent report has revealed that peroxisome proliferator-activated receptor-gamma (PPARgamma) is expressed not only in adipocytes but also in monocytes/macrophages and has suggested that PPARgamma may have a role in the differentiation of monocytes/macrophages. Furthermore, 9- and 13-hydroxy-(S)-10,12-octadecadienoic acid (9- and 13-HODE, respectively), the components of Ox-LDL, may be PPARgamma ligands. Therefore, we investigated the involvement of PPARgamma in the regulation of VEGF by Ox-LDL. PPARgamma expression was detected in human monocyte/macrophage cell lines, human acute monocytic leukemia (THP-1) cells, and human coronary artery endothelial cells (HCAECs). Ox-LDL (10 to 50 microg/mL) upregulated VEGF secretion from THP-1 dose-dependently. VEGF mRNA expression in HCAECs was also upregulated by Ox-LDL. The mRNA expression of VEGF in THP-1 cells and HCAECs was also augmented by PPARgamma activators, troglitazone (TRO), and 15-deoxy-(12,14)-prostaglandin J(2) (PGJ2). In contrast, VEGF expression in another monocyte/macrophage cell line, human histiocytic lymphoma cells (U937), which lacks PPARgamma expression, was not augmented by TRO or PGJ2. We established the U937 cell line, which permanently expresses PPARgamma (U937T). TRO and Ox-LDL augmented VEGF expression in U937T. In addition, VEGF production by THP-1 cells was significantly increased by exposure to 9-HODE and 13-HODE. In conclusion, Ox-LDL upregulates VEGF expression in macrophages and endothelial cells, at least in part, through the activation of PPARgamma.

We have examined whether oxidized low-density lipoprotein (ox-LDL) affects the function of tissue-factor-pathway inhibitor (TFPI), an anti-coagulant regulator in the extrinsic pathway of coagulation, in cultured human umbilical vein endothelial cells (HUVEC). Treatment of culture medium of HUVEC with ox-LDL, but not with native or acetylated LDLs, drastically decreased the reactivity of TFPI to its antibody specific for Kunitz domain 1 or one specific for the conformation between Kunitz 1 and 2 of TFPI, and caused a rapid, concentration-dependent decrease in the functional activity of TFPI to inhibit Factor X activation. When 5 ng of recombinant TFPI (rTFPI) was mixed with 10 microg of ox-LDL for 30 min, almost all of the rTFPI was detected in the ox-LDL fraction and no free rTFPI was observed on non-denaturing PAGE, in contrast with the virtual absence of rTFPI in the native LDL fraction. Ox-LDL decreased the antigen level of TFPI in the lysate of HUVEC in a time-dependent manner. It did not affect the mRNA level, but ox-LDL-dependent reduction of the TFPI antigen level in HUVEC was reversed by the simultaneous treatment of ox-LDL with bafilomycin A1, an inhibitor of the lysosomal proton pump. These results indicate that ox-LDL lessens the anti-coagulant function of TFPI through both oxidative modification and accelerated degradation of the molecule outside and inside HUVEC respectively.

Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.

Background: It is unknown whether LDL particle size is, independent of other lipids and lipoproteins, associated with endothelial dysfunction in vivo., Methods and Results: We determined in vivo endothelial function in 34 healthy men by measuring forearm blood flow responses to intrabrachial artery infusions of acetylcholine (ACh, an endothelium-dependent vasodilator) and sodium nitroprusside (an endothelium-independent vasodilator). LDL peak particle size was measured with gradient gel electrophoresis. Men with small LDL particles (LDL diameter 25. 5 nm, n=24, blood flow 6.9+/-3.6 versus 11.4+/-5.1 mL/dL. min, P=0. 006). The groups had comparable LDL cholesterol concentrations (3. 9+/-0.6 versus 3.7+/-1.0 mmol/L, men with small versus large LDL particles), blood pressure, glucose concentrations, and body mass indexes. LDL size (r=0.45, P=0.01) but not HDL cholesterol (r=0.31, P=0.09) or triglycerides (r=-0.19, P=0.30) was significantly correlated with endothelium-dependent vasodilation. Serum triglyceride concentrations and LDL size were inversely correlated (r=-0.44, P=0.01). In multivariate regression analysis, LDL size was the only significant determinant of the ACh-induced increase in blood flow. Sodium nitroprusside-stimulated endothelium-independent vasodilation was similar in both groups., Conclusions: Small LDL particles are associated with impaired in vivo endothelial function independent of HDL and LDL cholesterol and triglyceride concentrations. LDL size may therefore mediate adverse effects of hypertriglyceridemia on vascular function.

Asymmetrical dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor. It is formed by protein arginine N-methyltransferases (PRMTs), which utilize S-adenosylmethionine as methyl group donor. ADMA plasma concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and producing proatherogenic changes of endothelial cell function. Four different isoforms of human PRMTs have been identified. Because the release of ADMA from human endothelial cells is increased in the presence of native or oxidized LDL cholesterol, we investigated the potential involvement of PRMT activity and gene expression in this effect. We found that the production of ADMA by human endothelial cells is upregulated in the presence of methionine or homocysteine and inhibited by either of the methyltransferase inhibitors S-adenosylhomocysteine, adenosine dialdehyde, or cycloleucine. This effect is specific for ADMA but not symmetrical dimethylarginine. The upregulation of ADMA release by native and oxidized LDL is abolished by S-adenosylhomocysteine and by the antioxidant pyrrollidine dithiocarbamate. Furthermore, a methyl-(14)C label is transferred from S-adenosylmethionine to ADMA but not symmetrical dimethylarginine, in human endothelial cells. The expression of PRMTs is upregulated in the presence of native or oxidized LDL. Our data suggest that the production of ADMA by human endothelial cells is regulated by S-adenosylmethionine-dependent methyltransferases. This activity is upregulated by LDL cholesterol, which may be due in part to the enhanced gene expression of PRMTs. In concentrations reached by stimulation of methyltransferases (5 to 50 micromol/L), ADMA significantly inhibited the formation of (15)N-nitrite from L-[guanidino-(15)N(2)]arginine. These findings suggest a novel mechanism by which ADMA concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and atherosclerosis.

Objective: To understand the role of oxidized low density lipoprotein (OX-LDL) in the pathogenesis of thrombotic complications in atherogenesis., Methods: Low density lipoprotein was isolated from normal heparinized blood by density gradient ultracentrifugation and oxidized by CuCl2. Total RNA was extracted from human umbilical vein endothelial cells (HUVECs) exposed to LDL or OX-LDL, using the guanidinium isothiocyanate method. The quantification of tissue factor pathway inhibitor (TFPI) mRNA in HUVECs was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR)., Results: HUVECs were able to express TFPI mRNA constitutively. The expression was not affected by LDL but was effectively inhibited by OX-LDL in a time- and dose-dependent manner., Conclusions: The results suggest that oxidized LDL may play an important role in inducing coagulation in atherosclerotic lesions by the inhibition of expression of TFPI in vascular endothelial cells.

Background: We have recently demonstrated a lectin-like receptor for oxidized (ox)-LDL (LOX-1) in human coronary artery endothelial cells (HCAECs). This receptor is upregulated by ox-LDL. The present study examined the significance of LOX-1 in monocyte adhesion to HCAECs and endothelial injury in response to ox-LDL., Methods and Results: HCAECs were incubated in the presence of antisense oligodeoxynucleotides to the 5'-coding sequence of the human LOX-1 gene (0.5 microm/L). Basal LOX-1 mRNA and protein were suppressed by antisense LOX-1. Ox-LDL-mediated upregulation of LOX-1 was also suppressed by antisense LOX-1. Incubation of HCAECs with ox-LDL (40 microg/mL) for 24 hours markedly increased monocyte chemoattractant protein-1 (MCP-1) mRNA and protein expression as well as monocyte adhesion to HCAECs (P<0.01). After 48 hours of preincubation of HCAECs with antisense LOX-1, ox-LDL-mediated upregulation of MCP-1 and monocyte adhesion to HCAECs both were suppressed (P<0.01), whereas sense LOX-1 had no effect. Whereas antisense or sense LOX-1 alone (both 0.5 nmol/L) did not injure the cells, antisense LOX-1, but not sense LOX-1, reduced ox-LDL-mediated HCAEC injury, determined as LDH release (P<0.01). Activation of mitogen-activated protein kinase (MAPK) may play a critical role in signal transduction in ox-LDL-mediated alteration in MCP-1 expression, since antisense LOX-1, but not the sense LOX-1, completely inhibited the ox-LDL-induced MAPK activation., Conclusions: These observations with the first use of a specific antisense to human LOX-1 mRNA suggest that LOX-1 is a key factor in ox-LDL-mediated monocyte adhesion to HCAECs.

Atherosclerosis is absent in human intramyocardial (buried) coronary arteries but atherosclerosis may be severe in superficial segments of the same vessels. The development of atherosclerosis has three phases: a plasma phase, a transfer phase and an intramural phase. The transfer phase involves the transfer of low-density lipoproteins and macrophages from the plasma into the arterial wall. Efficiency of transfer is low where plasma flow near the wall is rapid. Eddy currents caused by arterial branches produce low flow near the arterial wall. Plasma recalculates and moves slowly in these eddy currents and thus prolongs contact of LDL and macrophages with the wall, increasing the occurrence of atherosclerosis. Absence of atherosclerosis in buried vessels appears due to the effects of myocardial contraction on the transfer phase. Contraction of the myocardium surrounding buried arterial vessel compresses these vessels and moves the plasma, LDL and macrophages away from the wall. This will decrease transfer into the wall and act to prevent the development of atherosclerosis. Similar but less striking effects occur where bridges of myocardium cross arterial vessels. Possible applications to human disease are discussed briefly.

We investigated the effects of two well established risk factors for cardiovascular disease, homocysteine and oxidized low density lipoprotein (ox-LDL), on endothelial cell thrombogenicity. For this purpose we studied platelet adhesion to human endothelial cells (EC) under flow conditions at a shear rate of 350 s(-1) following EC treatment with either homocysteine or ox-LDL. Treatment of EC with either homocysteine (1 or 10 mmol/L for 16 h) or ox-LDL (100 microg/ml for 16 h) resulted in a 2-3 fold enhancement in platelet adhesion. The enhancement in platelet adhesion induced by 1 mmol/L homocysteine, but not that induced by 10 mmol/L homocysteine, was absolutely dependent on fibrin formation. Homocysteine treatment has significantly increased the cell surface tissue factor (TF) activity and slightly reduced the expression of the intercellular adhesion molecule I (ICAM-1). In contrast, ox-LDL treatment upregulated ICAM-1 expression and had no significant effect on endothelial TF activity. Neither homocysteine nor Ox-LDL affected surface expression of the alpha(v)beta3 integrin. The homocysteine-induced enhancement in platelet adhesion was almost completely abolished by blockade of the EC TF activity by a polyclonal antibody. The enhancing effect of homocysteine was also greatly reduced by inhibition of the EC alpha(v)beta3 integrin, but was not affected by blockade of EC ICAM-1. On the other hand, ox-LDL-induced enhancement in platelet - EC adhesion was greatly inhibited by blocking ICAM-1 or alpha(v)beta3, but remained unaffected by inhibition of TF activity. Preincubation of platelets with the glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist Reo-Pro has virtually abolished the enhancing effect of both homocysteine and ox-LDL. Our results suggest that homocysteine and ox-LDL might increase endothelial thrombogenicity by distinct mechanisms: homocysteine - by inducing TF activity, and ox-LDL - by upregulating ICAM-1, both of which enhance GPIIb-IIIa/fibrinogen dependent platelet adhesion to EC. The alpha(v)beta3 integrin, although not affected by EC stimulation, seems to play a crucial role in platelet-EC interaction regardless of the mechanism of EC perturbation.

Unlabelled: Progestins oppose the effects of estrogens in many biological systems, but it is not known if progestins oppose the antioxidant effects of estrogen and to differing degrees. To test these questions, the effects of various sex steroids on LDL oxidation and cytotoxicity were studied in the absence or presence of endothelial cells. Freshly isolated LDL was incubated in the presence of Cu(++) in the absence or presence of cultured bovine aortic endothelial cells in phenol red-free medium and without or with hormones in 0.5% ethanol. The hormones included 17beta-estradiol (E(2)), progesterone (Pg), norgestimate (NGM), levonorgestrel (LNG), and medroxyprogesterone acetate (MPA). LDL oxidation was measured as formation of conjugated dienes, lipid peroxides, and TBARS, and cyotoxicity by tetrazolium salt reduction (MTT reduction). Progestins diminished conjugated diene lag phase, accelerated lipid peroxide and TBARS production in the absence and presence of cells and accelerated cytotoxicity. When E(2) and progestin were incubated with cells at a molar ratio of 1:5, lipid peroxides were reduced from baseline by E(2) alone 31%, E(2)/Pg 29%, E(2)/NGM 16%, E(2)/LNG 9% (all P<0.05 or more) and E(2)/MPA 8% (ns) (E(2) or E(2)E(2)/NGM, E(2)/LNG and E(2)/MPA [P<0.001]; E(2)E(2)/LNG or E(2)/MPA [P<0.05]). MTT reduction followed a similar gradient, greatest with E(2) alone, least with E(2)/MPA., Conclusions: Progestins promote LDL oxidation and, conjointly, endothelial cell cytotoxicity. Progestins oppose the antioxidant and cytoprotective effects of estrogen when given in combination. MPA and LNG have the strongest prooxidant and cytotoxic effects, which may limit the cardiovascular benefit of estrogen during combined administration in vivo.

Elevated plasma level of lipoprotein(a) (Lp(a)) is a well established risk factor for premature atherosclerosis and coronary artery disease. Recent studies showed impaired endothelium-dependent vasodilatation in humans with elevated plasma Lp(a). However, these human studies could not determine whether (1) elevated Lp(a) levels alone are the cause of endothelial dysfunction (these patients had multiple risk factors), and (2) native or oxidatively modified Lp(a) contributes to endothelial dysfunction (no measurements of native/oxidized Lp(a) ratio was reported in humans). In order to test whether apo(a) (an essential component of Lp(a) which is required for binding to endothelial cells) and native Lp(a) cause endothelial dysfunction, in the present study we tested endothelium-dependent vasorelaxation in aortic rings isolated from control and transgenic male mice either expressing the human apo(a) gene (TgA) or both the human apo(a) and human apo B100 genes (TgL). The TgA mice had plasma apo(a) levels of 8.8 +/- 1.2 mg/dl (n=6) and the double transgenic TgL mice had plasma Lp(a) levels of 15.3 +/- 1.4 mg/dl (n=8). Isolated aortic rings with and without endothelium were mounted in organ chambers and contracted with U46619 (10(-8) M) in the presence of ibuprofen (10(-5) M). Acetylcholine caused concentration-dependent (10(-9)-10(-5) M) relaxation, which could be prevented by endothelium removal and by NG-L-nitro-arginine (10(-4) M). Basal and acetylcholine-stimulated endothelium-dependent relaxation and endothelium-independent relaxation to nitroglycerin (10(-6) M) were not significantly different in aortic rings isolated from control and TgA or TgL mice. Twenty-four hour incubation of aortic rings isolated from control mice with recombinant human apo(a) or native Lp(a) (up to 300 microg/ml) caused no impairment of endothelium-dependent relaxations. In contrast, incubation with oxidized Lp(a) (50 microg/ml) or oxidized LDL (250 microg/ml) caused significant suppression of acetylcholine-induced endothelium-dependent vasorelaxation. These results show for the first time that elevated plasma levels of apo(a) and Lp(a) do not cause endothelial dysfunction in transgenic mice.

Atherogenic lipids, particularly oxidized low-density lipoprotein, are responsible for a wide range of cellular dysfunctions within the vessel wall. The effects on endothelial cells disrupt normal control of vasomotion, with a reduction of effective nitric oxide activity, the development of a procoagulant surface, chronic low-grade inflammation, and abnormal cell growth. These changes are central not only in the development of atherosclerosis but also in the evolution of both stable and unstable ischemic syndromes. There is growing evidence that these abnormal changes in cell function respond rapidly to changes in the atherogenic lipids. Certain cell functions can improve within hours or days of cholesterol lowering.

The authors present the evidence of atherogenic properties of VLDL and LDL potentiation on the model of endothelial cells-human umbilical vein endothelial cells, by preferable stimulation of the endothelial cell to thromboxane A1 production at in vitro conditions by atherogenic lipoproteins. The vasoconstrictive, thrombogenic and atherogenic effects of TXA2 are exerted on the vessel in this way. The ratio prostacycline/thromboxane, decisive for the maintenance of vascular homeostasis, is less than 1, this means the beneficial effect of prostacycline can not be applied. Protective, antiatherogenic effect of HDL and its subfractions HDL2 and HDL3/predominantly through their function in the reverse cholesterol transport from the periphery to the liver, antioxidative influence on LDL, as far as antiaggregation and fibrinolytic effects of HDL/is multiplied by the fact that HDL preferably stimulates the secretion of prostacycline by the endothelial cell. The ratio prostacycline/thromboxane A2 is higher than 1, that means beneficial vasodilative, antiaggregation and antiatherogenic effect of prostacycline on the vessel wall predominate. Quantitative evaluation of antiatherogenic effects of HDL subfractions (HDL2 and HDL3) revealed more significant antiatherogenic effect in HDL2 subfraction-in the sense of prostacycline secretion stimulation and exertion of its beneficial effects on the vessel. (Fig. 5, Ref. 33.)

The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.

Oxidized low-density lipoprotein (ox-LDL) plays a critical role in the development of atherosclerosis. Recent studies show that ox-LDL may induce apoptosis of cultured rabbit smooth muscle cells and human macrophages. This study was designed to determine the modulation by ox-LDL of apoptosis in cultured human coronary arterial endothelial cells (HCAEC) during hypoxia-reoxygenation and to determine underlying mechanisms. When HCAEC were approximately 85% confluent, the cells were exposed to hypoxia (24 h)-reoxygenation (3 h), native LDL, or ox-LDL. Fragmented DNA end-labeling, DNA laddering, and light and electron microscopy were used to determine changes characteristic of apoptosis. Ox-LDL (20 microg/ml) increased apoptosis during hypoxia-reoxygenation compared with hypoxia-reoxygenation alone (P < 0.05). Low concentrations of ox-LDL (5 microg/ml) and native LDL (20 microg/ml) under identical conditions had no effect on the degree of apoptosis. Ox-LDL markedly decreased endogenous superoxide dismutase activity and increased lipid peroxidation in HCAEC. The presence of ox-LDL, but not native LDL, in cultured HCAEC resulted in the activation of protein kinase C (PKC) and protein tyrosine kinase (PTK). The specific PKC and PTK inhibitors significantly reduced ox-LDL-mediated apoptosis of HCAEC (P < 0.05). Hypoxia-reoxygenation significantly increased Fas expression and decreased bcl-2 expression in HCAEC lysate as determined by Western analysis. Ox-LDL further increased Fas expression and decreased bcl-2 expression. These data indicate that ox-LDL enhances hypoxia-reoxygenation-mediated apoptosis in HCAEC. Ox-LDL-mediated apoptosis of HCAEC appears to involve activation of PKC and PTK. In addition, ox-LDL modulates Fas and bcl-2 protein expression in HCAEC. This study also suggests that ox-LDL is more important than native LDL in hypoxia-reoxygenation-induced apoptosis.

Treatment of human umbilical vein endothelial cells (HUVEC) with oxidized low density lipoprotein (ox-LDL, 100 microg/ml) for 24 h increased adhesion of human monocytic Mono Mac 6 cells from 4.8 +/- 0.9% to 17.6 +/- 2.5% (P < 0.001). The effect was dose dependent and first evident at 10 microg/ml ox-LDL. In contrast, adhesion of U937 cells was not significantly increased. Mac-1 (CD11b/CD18), a monocytic counter-receptor for intercellular adhesion molecule-1 (ICAM-1), that also binds to heparin, is present on Mono Mac 6 but not on U937 cells, and may thus explain these differences in adhesion. Consistently, ox-LDL induced a 2-fold upregulation of ICAM-1 surface expression on HUVEC. The presence of maltose-1-phosphate or heparin but not monoclonal antibodies (mAbs) to ICAM-1 reduced adhesion of Mono Mac 6 cells to untreated HUVEC. Combinations of mAbs to ICAM-1 with either maltose-1-phosphate or heparin inhibited Mono Mac 6 adhesion to ox-LDL-stimulated HUVEC by more than 50%, while either alone had no effect. This suggests that two distinct endothelial ligands for Mac-1, inducible ICAM-1 and carbohydrate-decorated heparin-like proteoglycan structures mediate monocytic cell interaction with ox-LDL-treated HUVEC. The stimulating activity in ox-LDL could partly be transfered to bovine serum albumin, while lysophosphatidylcholine or 8-epi prostaglandin F2alpha produced no stimulatory effects. The inhibition of ox-LDL effects with the antioxidant PDTC indicates radicals as possible mediators. In conclusion, we show that oxidatively modified LDL induces adhesion of monocytic cells, which utilize at least two distinct adhesive receptors on endothelium, one being identified as ICAM-1.

We present evidence that hypercholesterolemia and oxidized low-density lipoprotein (ox-LDL) impair endothelial cell growth by suppressing basic fibroblast growth factor (bFGF) expression. Background studies show that diet-induced hypercholesterolemia in rabbits impairs hyperplastic lumen-expanding remodeling of the carotid artery in response to a chronic flow load. Hypercholesterolemia also markedly impairs compensatory macrovascular and microvascular growth in rabbit ears with surgical restriction of arterial supply. In an in vitro model of angiogenesis, arterial explants cultured in a three-dimensional collagen gel exhibited organized endothelial cell growth with formation of capillary-like microtubes (CLM). CLM growth was sensitive to inhibition by neutralizing antibodies against bFGF. With explants excised from both the aorta of hypercholesterolemic rabbits and from coronary arteries of patients with coronary arteriosclerosis, CLM growth and release of immunoassayable bFGF to the culture medium were suppressed. Growth suppression was reversed partially by exogenous bFGF. In control explants, ox-LDL produced a suppression of CLM growth that could be reversed by exogenous bFGF. In endothelial cells in culture, ox-LDL suppressed bFGF expression and DNA synthesis in a dose-dependent manner. We conclude that atherosclerosis is associated with impaired bFGF-dependent endothelial cell growth manifested by impaired adaptive growth responses of large arteries and microvessels.