The various effects of fractionated oxidized low density lipoproteins on the growth of smooth muscle cells in culture

The effects of fractionated oxidized low density lipoproteins (oxidized LDL) on the growth of vascular smooth muscle cells (VSMC) and their relationship to the formation of lysophosphatidylcholine (lyso-PC) as well as the activation of protein kinase C (PKC) were studied. VSMC were isolated from porcine aorta by explant culture. LDL was isolated from porcine blood by sequential ultracentrifugation and oxidized LDL was obtained by incubating LDL with 5 µM CuSO₄ at 37° C for various lengths of time. Our results showed that LDL oxidized for 12 h and eluted from fast protein liquid chromatography at 43 min inhibited the growth of VSMC, and that LDL oxidized for longer than 48 h and eluted at 48 min stimulated the growth of VSMC. The formation of lyso-PC in the oxidized LDL correlated well with its stimulatory effect, suggesting that lyso-PC is responsible for the mitogenic effect of oxidized LDL. This stimulatory effect of oxidized LDL was inhibited by staurosporine, a PKC inhibitor. Treatment with oxidized LDL increased the activity of membrane PKC, but it decreased that of cytosolic PKC, suggesting the translocation of PKC from cytosol to the membrane in the presence of oxidized LDL. These results suggested that the oxidized LDL-stimulated VSMC growth was mediated by the formation of lyso-PC and the activation of PKC.     

Effects of oxidative modification of cholesterol in isolated low density lipoproteins on cultured smooth muscle cells

Summary It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can participate in atherosclerosis. The present paper studied the effect of cholesterol oxidation in LDL on cultured vascular smooth muscle cells. LDL was oxidized by cholesterol oxidase (3--hydroxy-steroid oxidase) which catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. Cholesterol oxidase treatment of LDL did not result in lipid peroxidation. Cultured rabbit aortic smooth muscle cells were morphologically changed following exposure to cholesterol oxidized LDL. Nile red, a hydrophobic probe which can selectively stain intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with oxidized or non-oxidized LDL cholesterol. LDL which did not undergo oxidation of its cholesterol had no effect on the cells. However, cellular nile red fluorescence intensity was increased as the pre-incubation time of cholesterol oxidase with LDL increased. This was supported by HPLC analysis which revealed that the oxidized cholesterol content of treated cells increased. These findings suggest that cholesterol oxidation of LDL can alter lipid deposition in the cells and change cell morphology. The oxidation of cholesterol in vivo may play an important role in the modification of LDL which could contribute to the generation of the lipid-laden foam cells.     

Endogenous arachidonic acid metabolism by cultured arterial smooth muscle cells

Cultured aortic smooth muscle cells originated from healthy and atherosclerotic rabbits produce prostaglandins (namely prostacyclin) at a basal state. Prostaglandin secretion is dramatically reduced in atherosclerotic cells. This impairment was not correlated with any alteration of acyl hydrolase activities and probably involved a decrease of cyclooxygenase activities.     

Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation

The effects of oxidized human plasma low density lipoproteins (Ox-LDL) on the proliferation of cultured aortic smooth muscle cells was studied, employing viable cell counting, [³H] thymidine incorporation into DNA, and the release of lactate dehydrogenase (LDH) into the medium. Oxidized LDL (prepared by incubation of LDL with copper sulfate) exerted a concentration-dependent stimulation (2 fold, compared to control) of aortic smooth muscle cell proliferation at low concentrations (0.1 µg – 10 µg/ml medium). On the other hand, at high concentrations (25–200 µg/ml), Ox-LDL produced a pronounced decrease in viable cells, a decrease in the incorporation of [³H] thymidine into DNA, and an increase in the release of LDH in the medium. In this report, the previously postulated biological roles of oxidized-LDL in atherosclerosis are discussed in view of these findings.Abbreviations Ox-LDL Oxidized human plasma Low Density Lipoproteins - SMC Smooth Muscle Cells - LDH Lactate Dehydrogenase - LPC Lysophosphatidycholine - PC Phosphatidylcholine - TNF Tumor Necrosis Factor     

Acetylated low density lipoproteins promote the release and metabolism of arachidonic acid by murine macrophages

It has been postulated that the ratio of prostacyclin/thromboxane A2 in the blood is an important marker for atherosclerosis. We studied the role of the Acetylated Low Density Lipoprotein (Acetyl-LDL) on the arachidonic acid metabolism in macrophages, the progenitor of the foam-cells in atheroma. When stimulated by Acetyl-LDL, macrophage released and metabolized arachidonic acid. This effect was time- and dose-dependent. Only 50% of the Acetyl-LDL-induced arachidonic acid released was metabolized while more than 90% of zymosan or A23187 induced arachidonic acid released was metabolized. Furthermore, when the macrophages were stimulated by Acetyl-LDL, a decrease of prostaglandin E2 and an increase of the levels of prostacyclin and thromboxane were noted. The implications of these observations in the pathogenesis of atherosclerosis are discussed.     

Effects of oxysterols on arachidonic acid metabolism and prostacyclin synthesis in bovine aortic smooth muscle cells in culture

Previous studies have shown that oxysterols could induce arterial damage in animals and manifest potent toxicity in cultured cells. Bovine aortic smooth muscle cells in culture were used to study the effects of several cholesterol oxides on arachidonic acid (AA) metabolism. Using two different methods, i.e. radioactive labeling of cells with 14C-AA and radioimmunoassay of 6kPGF1 alpha, the stable metabolite of Prostacyclin (PGI2), we observed various effects depending on the substance. Oxysterols oxidised on the rings were able to inhibit AA metabolism only at high doses, toxic to the cells, presumably through a non specific lytic mechanism. Oxysterols oxidised on the side chain induced an inhibition of the overall arachidonate conversion and PGI2 synthesis at low doses, below the range of cytotoxicity. This inhibition was noted both on the basal and stimulated metabolism. Mechanisms involved in such actions are still to be determined.     

Increase in nuclear calcium in smooth muscle cells exposed to oxidized low density lipoprotein

Vascular smooth muscle cells respond with an increase in intracellular Ca²⁺ within seconds after exposure to oxidized low density lipoprotein (oxLDL). This has been suggested to represent a signaling response that may have implications for gene expression. If so, oxLDL may induce increases in nuclear Ca²⁺ in smooth muscle cells in response to oxLDL. Aortic smooth muscle cells were exposed to 100 μg/ml oxLDL. Large, rapid increases in [Ca²⁺]_i were observed using fluo-3 as an indicator dye to detect intracellular Ca²⁺ on the stage of a confocal micro-scope. This was also confirmed using ratiometric imaging of indo signals. These elevations appeared to be localized to the nuclear region of the cell. DNA staining of the cells confirmed its localization to the nuclear / perinuclear region of the cell. Our data demonstrate that oxLDL induces a nuclear localized elevation in Ca²⁺i that may have important implications for nuclear function.     

Increase in nuclear calcium in smooth muscle cells exposed to oxidized low density lipoprotein

Vascular smooth muscle cells respond with an increase in intracellular Ca²⁺ within seconds after exposure to oxidized low density lipoprotein (oxLDL). This has been suggested to represent a signaling response that may have implications for gene expression. If so, oxLDL may induce increases in nuclear Ca²⁺ in smooth muscle cells in response to oxLDL. Aortic smooth muscle cells were exposed to 100 μg/ml oxLDL. Large, rapid increases in [Ca²⁺]_i were observed using fluo-3 as an indicator dye to detect intracellular Ca²⁺ on the stage of a confocal micro-scope. This was also confirmed using ratiometric imaging of indo signals. These elevations appeared to be localized to the nuclear region of the cell. DNA staining of the cells confirmed its localization to the nuclear / perinuclear region of the cell. Our data demonstrate that oxLDL induces a nuclear localized elevation in Ca²⁺i that may have important implications for nuclear function.     

Synergistic interaction between thromboxane A2 and mildly oxidized low density lipoproteins on vascular smooth muscle cell proliferation

Low density lipoprotein (LDL) and mildly oxidized low density lipoprotein (mox-LDL) are known mitogens for vascular smooth muscle cell (VSMC). Since aggregating platelets at sites of atherosclerotic injury release thromboxane A2(TXA2), a known mitogen for VSMC, we examined whether TXA2 can act synergistically with mox-LDL or its oxidative components in inducing VSMC proliferation. Growth arrested primary aortic rabbit VSMCs in 1st or 2nd passage were incubated with different concentrations of LDL or mox-LDL or lysophosphatidylcholine (LPC) or H2O2 or 4-hydroxy-2-nonenel (HNE) for 24 h followed by incubation with TXA2 mimetic U46619 for another 24 h. The amount of 3[H]-thymidine incorporated into the DNA was measured. Both LDL and mox-LDL at a concentration of 120 microg/ml induced proliferation of VSMC (168% or 184% respectively) when compared to the control. U46619 induced VSMC proliferation was observed at a concentration of 5 microm/L. As compared to native LDL, the mitogenic effect of mox-LDL on VSMC proliferation was markedly potentiated by U46619 to 301% or 316% at 0.5 or 5 microm/L U46619 respectively. LPC, H2O2 and HNE induced DNA synthesis was also marked by enhanced by U46619. These results suggest that even low concentration of TXA2 released from aggregating platelets may potentiate the mitogenic effect of mox-LDL at sites of vascular damage. The mitogenic effect of mox-LDL may be mediated via its oxidation products LPC, H2O2 (reactive oxygen species donor), and HNE.     

Modified low density lipoproteins decrease the activity and expression of lysosomal acid lipase in human endothelial and smooth muscle cells

Lysosomal acid lipase (LAL), the only lysosomal enzyme involved in the hydrolysis of LDL-cholesteryl esters, is a key regulator of cellular cholesterol and fatty acid homeostasis and its deficiency contributes to the pathophysiology of various diseases. In this study, we questioned whether oxidized or glycated LDL, a common occurrence in atherosclerosis and diabetes, affect the activity and expression of LAL in vascular endothelial cells (EC) and smooth muscle cells (SMC). LAL activity and expression were assayed in cultured human EC and SMC exposed to oxidized LDL (oxLDL), (±)9-hydroxyoctadecadienoic acid-cholesteryl ester (HODE), glycated LDL (gLDL), or native LDL (nLDL) as control, in the presence or absence of LXR or PPAR-gamma agonists. We found that LAL activity and expression were significantly down regulated by oxLDL and HODE in EC, and by gLDL in SMC. The LXR agonist T0901317 reversed the decreased LAL expression in modified LDL- or HODE-exposed EC (P < 0.001) and in gLDL-exposed SMC, whereas PPAR-gamma agonist rosiglitazone induced a low effect only in EC. In conclusion, modified LDL down regulates LAL expression in human EC and SMC by a process involving the LXR signaling pathway. This is the first demonstration that modified LDL modulate LAL expression, in a cell specific manner.     

Overexpression of SERCA2 ATPaase in vascular smooth muscle cells treated with oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca²⁺ _i homeostasis and altered two Ca²⁺ release mechanisms in these cells (IP₃ and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca²⁺ pump. One of the primary Ca²⁺ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP₃ and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca²⁺ homeostasis in VSMC chronically exposed to atherogenic oxLDL.     

Lipoprotein lipase enhances the binding of native and oxidized low density lipoproteins to versican and biglycan synthesized by cultured arterial smooth muscle cells

Retention of low density lipoproteins (LDL) by vascular proteoglycans and their subsequent oxidation are important in atherogenesis. Lipoprotein lipase (LPL) can bind LDL and proteoglycans, although the effect of different proteoglycans to influence the ability of LPL to act as a bridge in the formation of LDL-proteoglycan complexes is unknown. Using an electrophoretic gel mobility shift assay, [(35)S]SO(4)-labeled versican and biglycan, two extracellular proteoglycans secreted by vascular cells, bound native LDL in a saturable fashion. The addition of bovine milk LPL dose-dependently increased the binding of native LDL to both versican and biglycan, approaching saturation at 30-40 microgram/ml LPL for versican and 20 microgram/ml LPL for biglycan. LDL was oxidized by several methods, including copper, 2, 2-azo-bis(2-amidinopropane)-2HCl and hypochlorite. Extensively copper- and hypochlorite-oxidized LDL bound poorly to versican and biglycan. Proteoglycan binding to LDL was correlated inversely with the extent of LDL; however, the addition of LPL to oxidized LDL together with biglycan or versican allowed the oxidized LDL to bind the proteoglycans in an LPL dose-dependent manner. Addition of LPL had a greater relative effect on the binding of extensively oxidized LDL to proteoglycans compared with native LDL. LPL had a slightly greater effect on increasing the binding of native and oxidized LDL to biglycan than versican. Thus, LPL in the artery wall might increase the atherogenicity of oxidized LDL, since it enables its binding to vascular biglycan and versican.     

Induction of growth-related metabolism in human vascular smooth muscle cells by low density lipoprotein

Human vascular smooth muscle cells (hVSMC) rendered quiescent by maintenance under serum-free culture conditions for 48 h exhibited several metabolic responses, normally associated with proliferation, following exposure to low density lipoprotein (LDL). LDL induced a time- and dose- (half-maximally effective concentration, ED50 25.0 +/- 8 nM) dependent activation of S6 kinase which could be negated following pretreatment of hVSMC with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 48 h. In myo-[3H]inositol-prelabeled hVSMC, LDL caused a rapid (maximum within 1 min) decrease in phosphatidylinositol 4,5-bisphosphate (35% p less than 0.001) and phosphatidylinositol 4-phosphate (20%, p less than 0.01) with a return to prestimulated levels within 5-10 min. LDL induced a concomitant increase in [3H]inositol phosphates for which the order of generation was inositol-tris greater than -bis greater than -mono phosphate and which reached threshold levels of significance (p less than 0.05) above control values within 1, 2, and 10 min, respectively. The effect of LDL on hVSMC phosphoinositide metabolism was dose-dependent (half-maximally effective concentration, ED50 32.1 +/- 5.0 nM). This concentration, like that for S6 kinase, approximates with the KD (5-21 nM) for high affinity binding of 125I-LDL to specific receptors (1.5 x 10(4) sites/cell) on hVSMC. LDL induced a rapid but transient translocation of protein kinase C from the cytosol to membranes as assessed using both immunoblotting and [3H] 4-beta-phorbol-12-13-dibutyrate-binding procedures. Exposure of quiescent hVSMC to LDL elevated intracellular pH (delta pH 0.30 +/- 0.03, p less than 0.001). Such alkalinization was prevented in the presence of Na+/K+ exchange inhibitors such as amiloride, dimethylamiloride, and ethylisopropylamiloride. In an investigation of the nuclear action of LDL, a time-dependent induction of both c-myc and c-fos was observed. Such LDL-induced expression of these nuclear proto-onco-genes was not detectable in protein kinase C down-regulated hVSMC. Nevertheless, in spite of the cascade of "growth-promotional" responses elicited by LDL in quiescent hVSMC, this lipoprotein alone (under serum-free conditions) was neither mitogenic in nuclear labeling experiments, nor could it support growth of hVSMC in culture. We demonstrate that LDL might function in a complementary/synergistic fashion with other weakly mitogenic (to VSMC) growth factors and suggest that activation of protein kinase C (vis à vis intrinsic tyrosine kinase characteristic of other growth factor receptors) may be crucial to the signal transduction pathway for LDL.     

Cholesterol-rich low density lipoproteins are also more oxidized

Cardiovascular diseases are accompanied by active oxygen species and organic free radical generation. The aim of this study was to examine the possibility of using oxidized low-density lipoprotein (oxLDL) as a new diagnostic biomarker. Epidemiological study in populations of Estonia (782 subjects) and Russia (1433 subjects) was carried out in 2007-2009. The screening procedure included standard epidemiological methods. Oxidative stress was assessed by measuring the level of oxLDL using immunoassay method. Positive correlation between the levels of oxLDL and LDL cholesterol was indicated in blood of patients from estonian (r = 0.61; P < 0.05) and russian (r = 0.56; P < 0.05) populations. In russian population oxLDL/HDL cholesterol ratio was higher in the groups with highest risk of atherosclerosis development or manifest coronary artery disease (CAD). Cholesterol-rich low density lipoproteins are also more oxidized. Estimation of oxLDL/HDL ratio may be used as an independent biochemical marker for atherosclerosis.     

Antibodies against oxidized low density lipoproteins in pregnant women

Oxidized low density lipoproteins (oxLDL) formed in vivo induce a humoral immune response. Oxidative modification of LDL renders it immunogenic and a heterogeneous population of specific anti-oxLDL antibodies is produced. These antibodies could represent a biological marker of oxidative stress and serve as markers of atherosclerosis. Autoantibodies against oxLDL (oLAb) have been detected in human subjects practically of every age. oLAb also appear in the blood of pregnant women. Some studies have shown that the levels of antibodies to oxLDL were elevated in women with established preeclampsia. The present study was aimed to estimate the oLAb IgG levels in the first and second trimester of pregnancy. Furthermore, we estimated the correlation between maternal serum (MS) levels of oLAb and alpha-1-fetoprotein (MS AFP), human chorionic gonadotrophin (MS HCG) and trophoblast-specific-beta-1-glycoprotein (MS SP1), because these proteins are determined as a part of prenatal biochemical screening for fetal congenital abnormalities. Our study deals with the oLAb changes in women with pregnancy-induced hypertension. We also investigated the correlation between oLAb IgG and anticardiolipin antibodies IgG (ACA) in the serum of pregnant women. We examined 40 pregnant women attending Institute for Mother and Child Care for their antenatal care as outpatients. Routine blood samplings between the 9-13th week of pregnancy and 16-18th week of pregnancy were performed as a part of biochemical prenatal screening for fetal congenital abnormalities (Group 1). Their mean age was 27 +/- 4.1 years. Furthermore, we examined 26 women in the second or third trimester with pregnancy-induced hypertension (Group 2). Group 2 was compared with 49 pregnant women in the second or third trimester who were normotensive (Group 3). We used commercial standardized ELISA kits for determination of oLAb IgG, ACA IgG, MS AFP and MS HCG, MS SP1 was analyzed by single radial immunodiffusion. We did not find any differences in the levels of oLAb IgG in the first and second trimester in the women of Group 1. The correlation between oLAb and ACA IgG was not statistically significant (Spearman coefficient r=0.22, p=0.1). The correlation between oLAb IgG with MS AFP, MS HCG and MS SP1 was not statistically significant. Weak negative correlation for AFP and HCG was suggested both in the first and in the second trimester. The levels of oLAb IgG in the group of women with pregnancy-induced hypertension were significantly lower than in the group of normotensive women (348 +/- 388 U/ml v.s. 579 +/- 400 mU/ml, p<0.01). We can conclude that the levels of oLAb do not differ in the first and second trimester of gravidity. However, we cannot exclude the possible influence of an inverse relationship between oLAb IgG titers and the synthesis of fetoplacental antigens. This finding is important especially in the context of the results of prenatal biochemical screening. Pregnancy-induced hypertension is associated with lower levels of oLAb. Weak cross-reactivity between oLAb and anticardiolipin antibodies may exist but there is a possibility that there are two different populations of antibodies reacting with various antigens.     

Transforming growth factor-beta up-regulates low density lipoprotein receptor-mediated cholesterol metabolism in vascular smooth muscle cells.

The effects of transforming growth factor-beta (TGF-beta) on low density lipoprotein (LDL) receptor-mediated cholesterol metabolism were evaluated in vascular smooth muscle cells. TGF-beta significantly increased the binding, uptake, and degradation of 125I-LDL. This increase was paralleled by an increase in LDL receptor mRNA steady state levels and an increase in cholesterol esterification. The increase in LDL cholesterol metabolism was independent of proliferation. LDL receptor expression in response to TGF-beta was not affected by coincubation with an antibody against platelet-derived growth factor or by cyclooxygenase inhibitors in arterial smooth muscle cells, suggesting that TGF-beta's effect was not mediated through platelet-derived growth factor or prostaglandins, as demonstrated in other cell systems. However, coincubation with pertussis toxin abrogated the effect of TGF-beta on LDL receptor expression, suggesting that a pertussis toxin-sensitive G-protein may be involved in the signal transduction pathway. These results are discussed in terms of their potential effects on cellular cholesterol trafficking.     

Cholesterol metabolism in pigeon aortic smooth muscle cells lacking a functional low density lipoprotein receptor pathway

Cholesterol metabolism was examined in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons that have been shown to lack a functional LDL receptor pathway. In cells incubated in the presence of whole serum or low density lipoprotein (LDL) the rate of cholesterol synthesis from [1-14C]acetate or of HMG-CoA reductase activity was 20-100 times greater than for mammalian cells incubated under the same conditions. Unexpectedly, cholesterol synthesis decreased by nearly 50% after preincubation for 24 hr with lipoprotein-deficient serum (LPDS). This occurred without a change in cellular cholesterol content. Neither the high rate of cholesterol synthesis nor the effect of LPDS could be accounted for by differences in cell turnover or state of growth. Cholesterol added in ethanol was ineffective in altering cellular cholesterol synthesis or esterification even though a near doubling in cellular free cholesterol content occurred. Cholesterol synthesis and esterification were, however, able to be regulated with 25-OH cholesterol and mevalonolactone, as indicated by their ability to suppress cholesterol synthesis and to stimulate cholesterol esterification. In spite of the high rate of endogenous cholesterol synthesis, cellular cholesterol content was maintained at a constant level by the efficient efflux of the newly synthesized cholesterol from the cell. Unlike mammalian cells that require a cholesterol acceptor in the medium for efflux to occur, cholesterol efflux from pigeon cells occurred in the absence of a cholesterol acceptor. This suggests either that pigeon cells utilize a different mechanism for cholesterol efflux or that they produce their own cholesterol acceptor. As a result of a lack of a functional LDL receptor pathway, pigeon smooth muscle cells do not maintain cholesterol homeostasis through the controlled uptake of exogenous LDL cholesterol, as do mammalian cells. Rather, pigeon smooth muscle cells would appear to regulate cholesterol concentrations at the level of either cholesterol synthesis or efflux.     

The effect of hypoxia on uptake and degradation of low density lipoproteins by cultured human arterial smooth muscle cells.

Atheroma have been produced in experimental animal by systemic hypoxia. This study assessed the effects of hypoxia on binding, uptake and degradation of human low density lipoprotein (LDL) by human arterial smooth muscle cells, the cell involved in atherogenesis. The LDL content of the smooth muscle cell grown in the usual conditions (95% air [20% O2], 5% CO2) increased with the incubation time of LDL in the medium (7.5 mug protein/ml of medium); the trypsin releasable LDL "binding" reached a plateau by 24 h (2.2 +/- 1.3 [x +/- S.D.]) ng/mug LDL protein added per 10(6) cells whereas the LDL in the cell after trypsinization ("net uptake") continued to increase up to 48 h (6.5 +/- 4.6 ng/mug LDL protein added per 10(6) cells at 48 h). LDL protein degradation increases rapidly between 7 and 48 h (10.4 ng/mug LDL protein added per 10(6) cells at 24 h) after an initial delay of approximately 7 h. Smooth muscle cells grown under hypoxic conditions (5%02) had similar LDL "binding " but showed increased "net uptake" (10.7 +/- 4.8 ng/mug LDL protein added per 10(6) cells) and a 36 +/- 13% decrease in degradation (p less than 0.05; n =8). The impaired degradation of lipoprotein by smooth muscle cells may, in part, explain the role of hypoxia in atherogenesis.