Oxysterol profiles of normal human arteries, fatty streaks and advanced lesions.

OBJECTIVE: Human atherosclerotic lesions of different stages have quantitative differences in cholesterol and oxysterol content, but information on the oxysterol profile in fatty streaks is limited. This study aims to provide more detailed oxysterol quantification in human fatty streaks, as well as normal aorta and advanced lesions. METHODS: A newly adapted method was used, including oxysterol purification by means of a silica cartridge; and it was ensured that artifactual oxysterol formation was kept to a minimum. Cholesterol and oxysterols were estimated by GC and identification confirmed by GC-MS in samples of normal human arterial intima, intima with near-confluent fatty streaks and advanced lesions, in necropsy samples. RESULTS: The oxysterols 7 alpha-hydroxycholesterol, cholesterol-5 beta, 6 beta-epoxide, cholesterol-5 alpha, 6 alpha-epoxide, 7 beta-hydroxycholesterol, 7-ketocholesterol and 27-hydroxycholesterol (formerly known as 26-hydroxycholesterol) were found in all the lesions, but were at most very low in the normal aorta, both when related to wet weight and when related to cholesterol. Most components of the normal artery showed some cross-correlation on linear regression analysis, but cross-correlations were weaker in the fatty streaks and advanced lesions. However, in fatty streak there was a marked positive correlation between 27-hydroxycholesterol and cholesterol. CONCLUSION: The findings confirm that oxysterols are present in fatty streaks and advanced lesions and may arise from different cholesterol oxidation mechanisms, including free radical-mediated oxidation and enzymatic oxidation.     

Disorders in the system of cyclic nucleotides in atherosclerosis: cyclic AMP and cyclic GMP content and activity of related enzymes in human aorta

Cyclic AMP and cyclic GMP content and activities of cyclic nucleotide metabolic enzymes were determined in intima and media of atherosclerotic and unaffected human aorta obtained shortly after death due to myocardial infarction. Cyclic AMP content in fatty streaks and atherosclerotic plaques was lower by three- and five-fold, respectively, as compared with uninvolved intima. Cyclic GMP level in atherosclerotic lesions was estimated to be three-fold higher than in grossly normal area. Basal activity of adenylate cyclase in fatty streaks and plaques was two- to six-fold lower than in unaffected intima. Besides, the ability of adenylate cyclase to be stimulated by the stable analogue of prostacyclin, carbacyclin, was suppressed in plaques. Guanylate cyclase activity in fatty streaks was 1.5- to three-fold higher than in normal tissue. The thiol-reducing agent, dithiothreitol, decreased the enzyme activity to normal level, suggesting the oxidative nature of guanylate cyclase activation in the lesion zone. There were no significant changes in cyclic AMP phosphodiestease activity in the regions of the atherosclerotic lesion. Cyclic GMP phosphodiesterase activity in atherosclerotic plaques was two-fold lower than in the intima of unaffected areas. We did not find differences in the content of cyclic nucleotides or related enzyme activities in the media of uninvolved areas of human aorta nor in the media underlying atherosclerotic lesions. Our findings suggest that development of human atherosclerotic lesions is accompanied by dramatic changes in the cyclic nucleotide metabolism featuring gradual hormonal receptor uncoupling from adenylate cyclase, activation of guanylate cyclase in fatty streaks and inhibition of cyclic GMP phosphodiesterase in plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

The content of lipoperoxidation products in normal and atherosclerotic human aorta.

To evaluate the role of lipid oxidation in atherogenesis the levels of lipid- and protein-bound products of peroxidation in normal and atherosclerotic areas of human aorta were investigated. The level of fluorescent (360/430 nm) lipid products was measured in chloroform-methanol extracts of aortic tissue. Normal intima, initial lesions and fatty streaks had a similar content of fluorescent substances. On the other hand, high level of fluorescent products was found in atherosclerotic plaques. Cholesterol covalently bound to proteins, which serve as a marker of lipoperoxidation, was measured by high performance liquid chromatography after mild alkaline hydrolysis of delipidated tissue protein samples. The levels of protein-bound cholesterol in initial lesions and fatty streaks were close to its content in uninvolved intima (59 +/- 18 and 92 +/- 18 vs. 70 +/- 13 nmol/g protein). The content of covalently bound cholesterol in atherosclerotic plaques was dramatically higher (90-fold) than in the normal tissue. In addition to protein-bound cholesterol, considerable amount of lipofuscin was revealed in the cells of atherosclerotic plaques, but not in the cells of normal intima, initial lesions or fatty streaks. Thus, the contents of all investigated lipid- and protein-bound products of lipoperoxidation in earlier atherosclerotic lesions were similar to their levels in normal tissue. It can be due to a low rate of oxidized product formation and/or high rate of its degradation in or elimination from the vessel wall.     

Cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2) exposed to cadmium chloride

To evaluate the role of lipid oxidation in atherogenesis the levels of lipid- and protein-bound products of peroxidation in normal and atherosclerotic areas of human aorta were investigated. The level of fluorescent (360/430 nm) lipid products was measured in chloroform-methanol extracts of aortic tissue. Normal intima, initial lesions and fatty streaks had a similar content of fluorescent substances. On the other hand, high level of fluorescent products was found in atherosclerotic plaques. Cholesterol covalently bound to proteins, which serve as a marker of lipoperoxidation, was measured by high performance liquid chromatography after mild alkaline hydrolysis of delipidated tissue protein samples. The levels of protein-bound cholesterol in initial lesions and fatty streaks were close to its content in uninvolved intima (59 ± 18 and 92 ± 18 vs 70 ± 13 nmol/g protein). The content of covalently bound cholesterol in atherosclerotic plaques was dramatically higher (90-fold) than in the normal tissue. In addition to protein-bound cholesterol, considerable amount of lipofuscin was revealed in the cells of atherosclerotic plaques, but not in the cells of normal intima, initial lesions or fatty streaks. Thus, the contents of all investigated lipid- and protein-bound products of lipoperoxidation in earlier atherosclerotic lesions were similar to their levels in normal tissue. It can be due to a low rate of oxidized product formation and/or high rate of its degradation in or elimination from the vessel wall.     

The content of cyclic nucleotides in an organ culture of normal and atherosclerosis-affected human aorta

Cyclic AMP and cyclic GMP content was measured in intima media of unaffected and atherosclerotic areas of human aorta in a short-term organ culture. It was demonstrated that during short-term cultivation the content of both cyclic nucleotides in tissues is constant. The cyclic AMP content in fatty streaks and atherosclerotic plaques is significantly (2 to 7-fold) lower than in unaffected intima. The cyclic GMP level in atherosclerotic lesions is 1.5 to 3-fold higher than in normal. The content of both cyclic nucleotides in the media underlying fatty streaks and atherosclerotic plaques is the same as in the normal tissue. The obtained data indicate serious disorders in the system of cyclic nucleotides during atherosclerosis.     

Characterization of low density lipoprotein-like particle in the human aorta from grossly normal and atherosclerotic regions.

Physical and chemical criteria of lipoproteins containing apolipoprotein B, extracted from human aortic intima, were compared with those of plasma low density lipoproteins (LDL). Homogenates of grossly normal intima and advanced atherosclerotic lesions were subjected to differential ultracentrifugation to isolate a d = 1.006--1.063 g/ml density fraction which was extensively characterized. By electroimmunoassay, over 90% of the recovered apolipoprotein B immunological reactivity was found in isolates from both plaques and normal intima. In isolates of plaque and normal intima, particles of the same size as LDL were found, although a small population of very large structures was also present in plaque fractions. Apolipoprotein composition was similar to that of plasma LDL except for the presence of human serum albumin in aortic isolates. Fractions from aorta demonstrated greater electrophoretic mobilities than LDL. The lipid composition of isolates from normal intima was similar to that of LDL. The lipid composition of plaque fractions showed a significant decrease in the cholesteryl ester to free cholesterol ratio and in the triglyceride content in comparison to LDL and to fractions from normal intima. The fatty acid pattern of the cholesteryl ester fraction from isolates of both normal and plaque aortic homogenates demonstrated a significant decrease in the linoleate to oleate ratio as compared to LDL. Our initial studies suggest that althought aortic fractions are similar to LDL by certain criteria, some differences observed are more pronounced in fractions from lesions than from normal intima.     

Fatty acids liberated from low-density lipoprotein trigger endothelial apoptosis via mitogen-activated protein kinases

Enzymatic modification of low-density lipoprotein (LDL) as it probably occurs in the arterial intima drastically increases its cytotoxicity, which could be relevant for the progression of atherosclerotic lesions. LDL was treated with a protease and cholesterylesterase to generate a derivative similar to lesional LDL, with a high content of free cholesterol and fatty acids. Exposure of endothelial cells to the enzymatically modified lipoprotein (E-LDL), but not to native or oxidized LDL, resulted in programmed cell death. Apoptosis was triggered by apoptosis signal-regulating kinase 1 dependent phosphorylation of p38. Depletion and reconstitution experiments identified free fatty acids (FFA) as the triggers of this pathway. Levels of FFA in native LDL are low and the lipoprotein is therefore not cytotoxic; enzymatic cleavage of cholesterylesters liberates FFA that can rapidly trigger an apoptosis signaling cascade in neighboring cells. Blockade of this pathway can rescue cells from death.     

3H-thymidine incorporation into human aorta cells in primary culture. An autoradiographic study

Primary cell culture has been obtained from intima and media of unaffected zones of human aorta and from atherosclerotic lesions (fatty infiltration, fatty streaks, atherosclerotic plaques). Cellular polymorphism was found in these cultures. Four morphological types of aortic cells are described: elongated, asymmetric, polygonal, and stellate cells. These cell types also differed in their proliferative activity. On the 7th day of culturing, polygonal cells do not incorporate 3H-thymidine; the thymidine index of other three cell types was similar. The thymidine index of medial cells was higher than that of intimal ones.     

Damage and regeneration of the endothelium of the aorta in experimental hypercholesterolemia

In dynamics of the experimental hypercholesterolemia in rabbits, peculiarities of endothelial regeneration have been studied. Comparison of proliferative activity level in endotheliocytes with structural-functional state of the endothelial monolayer at atherogenesis makes it possible to consider, that the lesion of the endothelium cannot be regarded as an initiating factor for formation of atherosclerotic lesions. Formation of the lesions in the internal lining of the arteries is preceded by certain disorders in permeability of the endothelial barrier at increasing concentration of cholesterin in blood plasma, accompanying with a sharp activation of the cell proliferative activity. When lipid plates and atherosclerotic plaques are already formed, the processes of the endothelial damage and regeneration occur in parallel. The regeneration is ensured with an intensive proliferation and growth of endotheliocytes onto deendotheliolized areas of the damaged intima.     

Fibronectin distribution in human aortic intima and atherosclerotic lesions: concentration of soluble and collagenase-releasable fractions

Fibronectin is associated with cell attachment and migration and interacts with fibrin, collagen and glycosaminoglycans; thus, it may be a factor in the focal proliferation of smooth muscle cells and collagen in atherosclerosis. We have measured, by rocket immunoelectrophoresis, the concentrations of soluble and collagenase-releasable fibronectin in normal human aortic intima and different types of atherosclerotic lesions. Soluble fibronectin concentration showed no significant difference between normal intima and lesions, but was 6-8-times higher than expected on the basis of plasma concentration and molecular mass. The concentration free in the interstitial fluid was about 3-times the expected level, suggesting that it originates from local synthesis as well as plasma insudation. In tissue, about half the fibronectin appeared to be reversibly associated with tissue components. Incubation with collagenase released fibronectin equal to twice the soluble fraction from normal intima and early proliferative lesions. In more advanced plaques that were accumulating lipid, the amount released was significantly higher (P less than 0.05) and more than 3-times the soluble fraction, suggesting that it might be involved in lipid accumulation. However, there was no correlation between release of fibronectin and bound low-density lipoprotein.     

Quantitative estimation of lipid-laden cells in atherosclerotic lesions of the human aorta.

The intima of the adult human aorta consists of three sublayers: a muscular layer lying next to the media, a median hyperplastic layer and an innermost connective tissue layer, adjoining the lumen. The cells inhabiting these sublayers were isolated by the method of alcoholic-alkaline dissociation from grossly normal areas, fatty streaks and atherosclerotic plaques. The populations obtained contained cells with different numbers of cytoplasmic inclusions and a number without any. In unaffected intima and in fatty streaks, the cells with lipid inclusions were found predominantly in the outermost intimal layer including the connective tissue and in part of the median hyperplastic layer. In the superficial layer of unaffected intima and the fatty streak, these cells accounted for 15 and 25% of the total cell population, respectively. In the plaque, most cells with lipid inclusions were localized in the median hyperplastic layer of the intima (10%). The muscular layer was characterized by the lowest content of cells with lipid inclusions both in the unaffected intima and atherosclerotic lesions (from 0.75% in unaffected intima to 5% plaques). Among the intimal smooth muscle cells of various shapes, the cells with lipid inclusions were most often found in the stellate cell subpopulation (5-35%). A possible role of stellate cells in atherogenesis is discussed.     

人动脉壁蛋白聚糖与血清脂蛋白形成的不溶性复合物

本文报道了人主动脉壁中正常及异常(脂纹,FS)区的三种蛋白聚糖(PG)即:硫酸软骨素PG(CSPG)、硫酸皮肤素—硫酸软骨素PG(DSCSPG)及硫酸乙酰肝素PG(HSPG)与血清极低密度脂蛋白(VLDL)及低密度脂蛋白(LDL)所形成的不溶性复合物。在30mmol/L Ca~(2+)对,三种PG都能与这两种脂蛋白形成不溶性复合物,随放置时间的增加,形成的复合物都发生解离,但其复合物形成的曲线及解离程度明显不同。DSCSPG与CSPG比较,前者与两种脂蛋白更易形成不溶性复合物且不易解离。HSPG与两种脂蛋白形成不溶性复合物所需时间远大于CSPG及DSCSPG。FS区及正常区三种PG形成复合物曲线类型相似,异常区CSPG、DSCSPG与VLDL形成的复合物量低于正常区的相应PG,而与VLDL则高于正常区的相应PG。异常区的HSPG与两种脂蛋白形成不溶性复合物的量均高于正常区。     

Participation of smooth muscle cells in the formation of atherosclerotic plaques]

A study of the participation of the smooth muscle cells in the formation of atherosclerotic lesions was made on the autopsy material with the use of specific antiserum to the smooth muscle actomyosin and of indirect Coons' method. Typical forms of atherosclerotic lesions in the aorta, cerebral vessels and coronary arteries were studied. Smooth muscle cells were detected in the thickened intima alongside the atherosclerotic lesions, in fatty streaks, in the fibrous tissue of the atherosclerotic plaque, but they were not found in the atheromatous masses. The proliferation and migration of the smooth muscle cells is regarded as an essential factor in the pathogenetic mechanisms of atherosclersis.     

Similarity Between Naturally Occurring Modified Desialylated, Electronegative and Aortic Low Density Lipoprotein

The sialic acid content of electronegative low density lipoprotein (LDL) and LDL isolated from human aortic intima was measured. Sialic acid level in electronegative LDL of healthy subjects was 1.7-fold lower than in native LDL. Sialic acid content in electronegative LDL of coronary atherosclerosis patients was 3-fold lower than in native LDL. Lipoproteins isolated from grossly normal human aortic intima and from fatty streaks contained 20-56% less sialic acid as compared to blood plasma LDL. A negative correlation was established between the ability of electronegative and aortic LDL to stimulate lipid accumulation in cells cultured from uninvolved human aortic intima and lipoprotein sialic acid content. The results obtained indicate that electronegative and aortic LDLs have a low sialic acid content, i.e., are desialylated lipoproteins. Considered together with the fact that all known atherogenic LDLs have similar characteristics, our findings suggest that modified LDLs are the same lipoprotein particles subjected to multiple modification.     

Morelloflavone, a biflavonoid inhibitor of migration-related kinases, ameliorates atherosclerosis in mice

While macrophages take up modified LDL to form foam cells and multiply to develop fatty streaks, vascular smooth muscle cells (VSMC) migrate from the media to intima, secrete extracellular matrix, and increase the volume of atherosclerotic lesions. A medicinal plant Garcinia dulcis has been used in traditional Thai medicine for centuries to treat various chronic human diseases. Morelloflavone, a biflavonoid and an active ingredient of the plant, has been shown to inhibit VSMC migration through its inhibition of multiple migration-related kinases such as focal adhesion kinase, c-Src, ERK, and RhoA. However, the exact role of morelloflavone in atherosclerogenesis was unknown. We fed Ldlr(-/-)Apobec1(-/-) mice with either normal chow or chow containing 0.003% morelloflavone for 8 mo and assessed the extent of atherosclerosis by the en face and cross-sectional analyses. A cell composition analysis of atherosclerotic tissue was carried out using immunohistochemical staining. Oral morelloflavone therapy significantly reduced the atherosclerotic areas of the mouse aortas (a 26% reduction), without changing plasma lipid profiles or weights. Immunohistochemical analyses showed that morelloflavone reduced the number of VSMC in the atherosclerotic lesion while it did not change the density of macrophages in the lesion or the percentages of proliferating and apoptotic cells. Oral, low-dose, morelloflavone therapy retards atherosclerogenesis by limiting the migration of VSMC into the intima in the mouse model of human atherosclerosis. Upon further investigation, morelloflavone may be found to be a novel oral antiatherosclerotic agent and a viable addition to the conventional therapies such as statins in humans.     

Activation of 15-lipoxygenase by low density lipoprotein in vascular endothelial cells. Relationship to the oxidative modification of low density lipoprotein.

Oxidatively-modified low density lipoprotein (LDL) is thought to play a significant role in the formation of lipid-laden macrophages, the primary cellular component of atherosclerotic fatty lesions. Recently, lipoxygenases have been implicated as a major enzymatic pathway involved in rabbit endothelial cell-mediated LDL modification. We investigated the effect of LDL on porcine aortic endothelial cell (PAEC) and human umbilical vein (HUVEC) and aortic endothelial cell (HAEC) lipoxygenase activity. By thin layer chromatography, we observed that human LDL stimulated the metabolism of radiolabeled arachidonic acid to 12 + 15-hydroxyeicosatetraenoic acid (HETE) in indomethacin-treated PAEC. Furthermore, radiolabeled linoleic acid, a specific substrate for the 15-lipoxygenase, was metabolized to its respective product 13-hydroxyoctadecadienoic acid (13-HODE) in the presence of LDL. Increased product formation in both studies was inhibited by the lipoxygenase blockers nordihydroguaiaretic acid (NDGA) and RG 6866. 15-HETE was confirmed as the predominant HETE product in LDL-treated cells by high performance liquid chromatography. Both porcine- and human-derived LDL stimulated the CL release of 15-HETE from cells as determined by radioimmunoassay. Release of immunoreactive 15-HETE was inhibited by NDGA, RG 6866, and 5,8,11,14-eicosatetraynoic acid (ETYA) but not by the selective 5-lipoxygenase inhibitor RG 5901. These lipoxygenase inhibitors had similar effects on the modification of LDL. Our results suggest that the oxidative modification of LDL by endothelial cells may be mediated in part through activation of 15-lipoxygenase.     

Variations with age in the topographic distribution of coronary atherosclerotic lesions as assessed by image analysis

The localization of macroscopic coronary atherosclerotic lesions was investigated for 184 cases by image analysis. Atherosclerotic lesions were classified into 2 types: fatty streaks and raised lesions. The overall distribution of fatty streaks in arteries involved only by fatty streaks and the distribution of raised lesions in those arteries having raised lesions were demonstrated at similar sites in 3 coronary vessels. If the localization of lesions was examined within age groups, those sites involved with fatty streaks in one age group were replaced with raised lesions in the next older age group; fatty streaks in this older group were located distally to the raised lesions. Subsequently the areas occupied with fatty streaks were replaced with raised lesions in the next older age group. Furthermore, raised lesions seldom appeared at the site where fatty streaks had not existed in the preceding age group. Similar changes in topographic distribution were present if such changes were analyzed according to extent of atherosclerosis. Data from cases with myocardial bridges show that both fatty streaks and raised lesions are seldom observed in the region distal to myocardial bridge. We conclude, thus, that fatty streaks are the precursors of raised lesions in the coronary artery.     

Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: important role of uric acid

Oxidized LDL is present within atherosclerotic lesions, demonstrating a failure of antioxidant protection. A normal human serum ultrafiltrate of Mr below 500 was prepared as a model for the low Mr components of interstitial fluid, and its effects on LDL oxidation were investigated. The ultrafiltrate (0.3%, v/v) was a potent antioxidant for native LDL, but was a strong prooxidant for mildly oxidized LDL when copper, but not a water-soluble azo initiator, was used to oxidize LDL. Adding a lipid hydroperoxide to native LDL induced the antioxidant to prooxidant switch of the ultrafiltrate. Uric acid was identified, using uricase and add-back experiments, as both the major antioxidant and prooxidant within the ultrafiltrate for LDL. The ultrafiltrate or uric acid rapidly reduced Cu2+ to Cu+. The reduction of Cu2+ to Cu+ may help to explain both the antioxidant and prooxidant effects observed. The decreased concentration of Cu2+ would inhibit tocopherol-mediated peroxidation in native LDL, and the generation of Cu+ would promote the rapid breakdown of lipid hydroperoxides in mildly oxidized LDL into lipid radicals. The net effect of the low Mr serum components would therefore depend on the preexisting levels of lipid hydroperoxides in LDL. These findings may help to explain why LDL oxidation occurs in atherosclerotic lesions in the presence of compounds that are usually considered to be antioxidants.     

Stellate cells of aortic intima: II. Arborization of intimal cells in culture.

The present study analyzed effects of different cAMP-elevators on cell morphology in primary culture of human intimal and medial cells from grossly normal and atherosclerotic areas. In primary culture of human aortic cells adenylate cyclase activator forskolin and other cAMP elevators induced arborization of cells, i.e. they reversibly changed the shape of cells. This resulted in the formation of thin branching processes and in the concentration of cytoplasm around the nucleus. In the culture, the shape of the arborized cells resembled that of stellate ones detected in the aortic intima in situ. The arborization of cells was accompanied by destruction of myofilaments. Due to cAMP elevators' effect, most of the arborized cells were exhibited in the cultures isolated from the elastic-hyperplastic layer of the intima. The number of arborized cells was significantly less in the cultures isolated from the musculo-elastic layer and still lesser in those isolated from media. We failed to reveal any significant difference in the number of arborized cells cultured from fatty streaks, atherosclerotic plaques and grossly normal aortic areas. Obtained results suggest that the previously revealed polymorphism of human aortic intimal cells may be accounted for by the cell shape transformations underlined by the mechanism similar to that of arborization in vitro.     

Endothelial calcium signaling in rabbit arteries and its local alterations in early-stage atherosclerosis

This study is to examine whether endothelial calcium signaling is different between atherosclerosis-prone thoracic aortas (TA) and atherosclerosis–resistant carotid arteries (CA) in normal rabbits and how it changes in early-stage atherosclerosis. Local endothelial calcium signaling was examined in arterial segments obtained from rabbits fed with normal or high-cholesterol diet for 1–4 weeks. Contrasting to normal CA, normal TA showed lower endothelial calcium signaling with more concentrated NF-κB in the endothelial nuclei. In the same hypercholesterolemic animal, fatty streak formation was much more prominent in TA than in CA. TA endothelial calcium signaling became augmented in the second week of hypercholesterolemia, being most pronounced in smooth regions adjacent to miniature fatty streaks. It was sporadically elevated even in regions away from any detectable TA fatty streak. When the entire TA was covered with fatty streaks in the fourth week of hypercholesteremia, endothelial calcium signaling returned to the original level. In comparison, CA endothelial calcium signaling was reduced around scattered fatty streaks. Reduced calcium signaling happened where CA fatty streaks were 150 μm long (covering 15–30 cells); and it extended to areas adjacent to larger fatty streaks. Moreover, NF-κB remained in the cytosol of endothelial cells covering CA fatty streaks. Our results indicate that inter-vascular differences in endothelial calcium signaling may provide partial explanation in their differential susceptibility in atherosclerosis.