Effect of deposited lipids in atheromatous lesions on the migration of vascular smooth muscle cells

In advanced atherosclerotic lesions, a decrease in smooth muscle cells is observed in the cap tissue. This causes the thinning of the cap, and may lead to plaque rupture. We studied the effect of deposited lipids on the migration of vascular smooth muscle cells, and identified the main cause of the effect. The lipids were extracted from atherosclerotic lesions in the human aorta at autopsy, and separated into three fractions with a Sep-Pak ODS cartridge. Then, each fraction was added to the lower part of a chemotaxis chamber, and cultured vascular smooth muscle cells to the upper part. After 4 hours incubation, the cells that had migrated to the opposite side were counted. The oxysterol-rich fraction (10 microg/ml) inhibited the migration, whereas the cholesterol ester and free cholesterol fractions did not. Finally, we tested the pure oxysterols, 7-ketocholesterol and 27-hydroxycholesterol. Both inhibited migration, whereas the free cholesterol and cholesterol ester did not. Oxysterols generated in the lipid pool might inhibit the migration of smooth muscle cells.     

Atherogenic mechanisms: enhancement of regression of atheromatous lesions by phthalazinol

Atherosclerosis was produced in rabbits by feeding 1% cholesterol pellets for 15 weeks to 35 male rabbits divided into two equal groups--the placebo control and phthalazinol group--with a comparable serum cholesterol level. The phthalazinol group was given 20 mg/kg of the compound daily. 13 rabbits of both groups were on 15 weeks of the treatment and the remaining 22 rabbits were treated for 30 weeks. The treated group exhibited a statistically significant increased removal of cholesterol from atherosclerotic aortas and improvement in pathological changes. This enhancing effect was more marked in the animals treated for 30 weeks. Such evidence indicates that there are substances capable of enhancing the removal of cholesterol from atheromatous lesions. In light of the pharmacological properties of phthalazinol, the possible role of thromboxane A2 released from platelets adhered and aggregated on atheromatous plaques, in the progression of atherosclerosis is worthy of continued investigation.     

LDL-apheresis; potential procedure for prevention and regression of atheromatous vascular lesion

Nine patients with familial hypercholesterolemia (FH), 6 with homozygotes and 3 with heterozygotes, were treated with long term repetitive LDL-apheresis. The techniques are simple plasma exchange with human albumin solution, double membrane filtration, and selective LDL-adsorption by dextran sulfate-cellulose gel. The average term was 3.5 years except for the two homozygotes for whom the treatment was only initiated in our facility. Plasma total cholesterol levels were controlled between pretreating level, 320 to 500 mg/dl, and posttreating level, 100 to 160 mg/dl, by biweekly treatments. All patients showed remarkable improvement of cutaneous and tendinous xanthomas. One homozygous patient died at 31 years old of myocardial infarction after 2 years of treatment. A homozygous patient who has been treated since 5 years old for 6 years was reexamined by angiography and was shown to have atheromatous lesions regressed in the aortic valve region and in the left renal artery.     

Long-term probucol treatment results in regression of xanthomas, but in progression of coronary atherosclerosis in a heterozygous patient with familial hypercholesterolemia

A 66-year-old male heterozygous familial hypercholesterolemia (FH) patient with significant coronary atherosclerosis has been treated by us with probucol (1000 mg daily) for eight years. This treatment has produced significant reductions in the cholesterol levels of his serum, low density lipoprotein (LDL), and high density lipoprotein (HDL) from 237 ± 20 mg/dl (mean ± S.D.) to 156 ± 15, from 175 ± 8 to 111 ± 16 mg/dl, and from 23 ± 4 to 19 ± 2 mg/dl, respectively. These reductions have been maintained for eight years. Serum triglyceride levels also decreased, from 220 ± 54 to 146 ± 36 mg/dl. During this period, marked regression of xanthomas on the eyelids and finger extensor tendons was observed, while thickness of the Achilles tendons was reduced from 21.0 mm to 13.0 mm. On the other hand, effort-induced anginal symptoms requiring additional antianginal medication have been noticed, and angiographically-demonstrated coronary atherosclerosis has progressed significantly during these eight years. These observations lead us to suggest that maintaining low levels of HDL cholesterol with probucol, even though resulting in satisfactory reduction of LDL cholesterol and marked regression of xanthomas, appears to be associated with the progression of atherosclerosis in the coronary arteries.     

Low density lipoprotein receptor activity in human monocyte-derived macrophages and its relation to atheromatous lesions.

Human peripheral monocytes, isolated from a previously unused source (a leukocyte concentrate byproduct of the plateletphoresis procedure for platelet transfusion), transformed into macrophages while cultured with 5% human serum or isolated lipoprotein fractions. Used for the study of their cholesterol (Chol) metabolism, these human monocyte-derived macrophages had a high-affinity receptor that is saturable, specific for low density lipoprotein (LDL) and maximally induced by incubation for as little as 24 hr in medium devoid of lipoproteins or Chol. Macrophages were shown to have separate receptor activity for 125I-labeled LDL and 125I-labeled acetylated LDL; macrophages that had been incubated with lipoprotein-depleted serum degraded native and acetylated LDL at similar rates. The receptor for LDL was functional in maintaining Chol homeostasis, as demonstrated by feedback inhibition of Chol synthesis after culture with LDL or very low density lipoprotein (VLDL). The Chol content of macrophages was doubled by incubation with medium containing VLDL compared to incubation with LDL (60 microgram of Chol per ml of medium). Incubation with native lipoproteins did not lead to accumulation of esterified Chol by macrophages. Changes in the Chol metabolism of macrophages, rather than modifications in circulating LDL, may cause these cells to store cholesteryl ester and take on the characteristics of cholesteryl ester-laden macrophages of atheromatous lesions.     

普罗布考对小鼠体内巨噬细胞胆固醇逆转运的影响及其作用机制

目的探讨普罗布考对小鼠体内巨噬细胞TC逆转运的影响及其作用机制。方法选择C57BL/6小鼠36只随机分为4组,给予不同剂量普罗布考(0,0.1%,0.5%,1.0%,分别为对照组,0.1%干预组,0.5%干预组,1.0%干预组),各组9只。添加饲料喂养6周后,腹腔注射乙酰化LDL与氚(3 H)-TC标记的RAW264.7小鼠巨噬细胞悬液,酶法测定血脂,用液闪计数仪测定血清、肝脏及粪便中3 H-TC占经腹腔注射3 H-TC的百分比。结果普罗布考干预6周后,与对照组比较,各干预组小鼠TC、HDL-C、LDL-C明显降低(P<0.05,P<0.01),肝脏和粪便中3 H-TC含量明显升高(P<0.05),血清3 H-TC含量明显降低(P<0.05)。各干预组HDL-C水平与3 H-TC排泄量呈负相关(r=-0.532,P<0.05)。结论普罗布考干预了小鼠体内巨噬细胞TC逆转运,加速了TC经粪便的清除,有利于动脉粥样硬化的防治。     

Effects of probucol on xanthomata regression in familial hypercholesterolemia

Fifty-one patients with familial hypercholesterolemia were treated for 2 to 4 years with probucol, cholestyramine, clofibrate and compactin in various combinations. Mean baseline serum cholesterol was 359 ± 10 mg/dl in the heterozygote, and 582 ± 52 mg/dl in the homozygote patients. We found that a combination of probucol, cholestyramine and compactin decreased serum cholesterol to normal or near normal in most of the heterozygote patients. In 3 severely affected heterozygote and all 8 homozygote patients, adequate cholesterol reduction was only possible with plasmapheresis plus a hypolipidemic agent.

Measurement of the Achilles tendon after 12 to 16 months of treatment showed that reductions in thickness occurred in all patients taking probucol, even in a single-drug regimen, in those undergoing plasmapheresis, especially if probucol was used and in those receiving a combination of cholestyramine and compactin. Probucol was most effective in patients who experienced the greatest decreases in high density lipoprotein (HDL) levels, whereas the cholestyramine-compactin combination worked without decreasing HDL concentrations. Combined clofibrate-cholestyramine therapy, by contrast, led to increased tendon thickness in all but 1 patient. It is believed that probucol exerts its positive effect on xanthomata regression by reducing the size of HDL particles, as was shown in this study. It has already been reported that smaller HDL particles are more active in reverse cholesterol transport. The direct peripheral action of probucol may have aided regression as well.     

Lipids deposited in human atheromatous lesions induce apoptosis of human vascular smooth muscle cells

To clarify whether lipids deposited in human atheromatous lesions induce apoptosis of vascular smooth muscle cells (SMC) and to identify the main component in deposited lipids responsible for inducing apoptosis, we examined the effect of lipids extracted from human atheromatous lesions on apoptosis of cultured SMC and analyzed the content of cholesterol in the lipids. When lipids extracted from atheromatous lesions were added to SMC, agarose electrophoresis of DNA showed a ladder pattern, DNA fragmentation assay detected an increase of fragmented DNA, and flow cytometric analysis demonstrated an increase of apoptotic cells. When the extracted lipids were fractionated by Sep-Pak ODS column, addition of the oxysterol-rich fraction to SMC resulted in a DNA ladder pattern and positive staining of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The oxysterol-rich fraction also increased fragmented DNA and apoptotic cells to a greater extent than the other two fractions. HPLC analysis showed that the quantity of 7-ketocholesterol in extracted lipids was large enough to induce SMC apoptosis. These results suggest that lipids deposited in human atheromatous lesions may induce apoptosis of SMC and that oxysterols may be important factor contributing to induce apoptosis among deposited lipids.     

Extracts of human atherosclerotic lesions can modify low density lipoproteins leading to enhanced uptake by macrophages

Plasma low density lipoproteins (LDL) and/or other lipoproteins containing apo B that accumulate in atherosclerotic lesions of human aortas exhibit structural changes that are associated with enhanced uptake in an unregulated fashion by macrophages in culture, resulting in the formation of foam cells in vitro. In an attempt to better characterize the structure-function modifications, we have incubated plasma LDL with extracts of human atherosclerotic plaques obtained at surgery, and determined whether such plaque-modified LDL also demonstrates enhanced uptake by cultured mouse peritoneal macrophages (MPM). Enhanced uptake was found which was linear over a concentration range of 100 micrograms lipoprotein protein/ml, as assessed by enhanced degradation of [125I]LDL and by stimulation of cholesterol esterification. Extracts of non-arterial human tissue were unable to induce this modification, suggesting tissue specificity. When delipidated apo B from tissue-treated [125I]LDL was subjected to SDS-PAGE, autoradiograms demonstrated, in addition to the B-100 band of apo B, a doublet of higher molecular weight than B-100 and a band just entering the gel, both at the expense of the B-100 band. No lower molecular weight bands suggestive of apo B degradation were seen. Modest increases in LDL electrophoretic mobility and thiobarbituric acid reactive substances were found following the incubation of LDL with plaque extracts. These changes could be inhibited by butylated hydroxytoluene (BHT), suggesting that free radical-induced lipid peroxidation was responsible for these modifications. However, since BHT did not inhibit the uptake of the tissue-incubated LDL by macrophages, the actual modification responsible for enhanced macrophage recognition did not appear to be free radical-induced. Uptake of plaque-modified [125I]LDL was inhibited by only 22% by a 20-fold excess of acetyl LDL or plaque-modified LDL. If the latter did not represent a mixture of modified and unmodified particles, this result would suggest that uptake was not mediated by the scavenger receptor. It is possible that foam cells are formed in vivo when LDL particles, which have been modified by interacting with components of the arterial wall, are taken up by tissue macrophages.     

普罗布考对高密度脂蛋白亚组分与抗氧化功能的影响

目的研究普罗布考对动脉粥样硬化(AS)兔HDL亚组分及氧化功能的影响,探讨普罗布考抗AS机制。方法选择18只新西兰大白兔随机分为:对照组6只,饲以普通饲料;AS组6只,饲以高脂饲料;普罗布考组6只,饲以高脂饲料基础上给予普罗布考400 mg/(kg·d)。1 2周后酶法检测血脂;分光光度计法检测血清对氧磷酶1(PON1)活性;采用化学沉淀法检测HDL_2/HDL;组织切片和HE染色检测主动脉内膜中层厚度(IMT)和动脉斑块/血管面积。结果实验12周后,与AS组比较,普罗布考组血清TC、LDL-C、HDL-C水平明显下降(P0.01);PON1活性明显升高(P=0.000);HDL_2/HDL明显降低(P=0.000),主动脉IMT明显减小(P=0.000),斑块面积/血管腔面积明显降低(P=0.002)。结论普罗布考通过提高PON1活性,降低HDL_2/HDL而改善HDL功能,减少主动脉IMT及斑块面积/血管腔面积,延缓AS进展。     

The effect of vitamin E, probucol, and lovastatin on oxidative status and aortic fatty lesions in hyperlipidemic-diabetic hamsters

Diabetes mellitus is associated with an increased risk of premature atherosclerosis, which may be due in part to an increased rate of low density lipoprotein (LDL) oxidation. Previous studies have shown that vitamin E, probucol, and lovastatin can reduce the oxidative susceptibility of LDL in normoglycemic animal models; however, few studies have investigated this in conjunction with aortic fatty streak lesion formation in diabetic hyperlipidemic models. Forty-eight Syrian hamsters were made diabetic by intraperitoneal injection of low dose streptozotocin. Diabetic animals (12 animals/groups) received a high saturated fat and cholesterol diet for 12.5 weeks. At 2.5 week of dietary treatments, the diet was supplemented with either: (1) 500 IU/day vitamin E (D+E); (2) 1% probucol w/w of the diet (D+P); (3) 25 mg/kg lovastatin (D+L); or (4) diabetic control (D). An age-matched group of hamsters (n=6) receiving the same diet but not made diabetic (ND) was used as control. At the end of the study, aortic arch foam cell-rich fatty streak lesion, plasma glucose, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), non-HDL-C, triglycerides (TG), phospholipids, alpha-tocopherol, plasma lipid peroxide and the susceptibility of LDL to copper-catalyzed oxidation were determined. Diabetes increased plasma glucose, and when combined with an atherogenic diet resulted in a further increase of plasma lipids. Vitamin E, probucol, and lovastatin significantly reduced plasma TG in the diabetic hamsters fed the atherogenic diet. Vitamin E treatment increased TC, probucol reduced HDL-C without affecting TC; whereas lovastatin reduced TC and selectively decreased non-HDL-C, and significantly reduced fatty streak lesion formation in the aortic arch. While vitamin E and probucol were effective in reducing several indices of oxidative stress including plasma lipid peroxides, cholesterol oxidation products and in vitro LDL oxidation, they had no effect on fatty streak lesion formation. Our results indicate that the LDL in diabetic animals is more susceptible to oxidation than in non-diabetic hamsters and that not only vitamin E and probucol but also lovastatin provide antioxidant protection. It appears that in this combined model of diabetes and hypercholesterolemia, progression of fatty streak lesion formation is mainly associated with changes in TC and non-HDL-C as affected by lovastatin, and is less dependent on the extent of LDL oxidation, changes in plasma TG level and oxidative stress status.     

Characterization of apoptotic macrophages in atheromatous tissue of humans and heritable hyperlipidemic rabbits.

Apoptotic macrophages are regularly found in atherosclerotic plaques indicating programmed cell death as one of their regulatory controls. The objective of this study was to characterize in more detail apoptotic macrophages in atherosclerotic lesions of humans and heritable hyperlipidemic (HHL) rabbits. Macrophages were immunohistochemically analyzed using antibodies directed against alphaMbeta2-integrins (CD11b/CD18), CD44, major histocompatibility complex (MHC) class I and II, inducible nitric oxide synthase (iNOS), manganese superoxide dismutase (MnSOD), tumor necrosis factor alpha (TNFalpha), p53, c-jun/AP-1 and rabbit macrophages (RAM-11) and the TUNEL (TdT-mediated dUTP nick end labeling) technique. Colocalization studies of human atherosclerotic carotid and aortic tissue showed apoptotic plaque macrophages also being MnSOD-, alphaMbeta2-integrin-, CD44-, MHC class I- and II-, iNOS-, TNFalpha- and p53-immunoreactive. Similar results occurred in atherosclerotic aortas of HHL rabbits. Computer-assisted morphometric analyses revealed a positive correlation of the area density of MnSOD-immunoreactive macrophages with those of alphaMbeta2-integrin- and CD44-immunoreactive ones, but not with those of MHC class I- and II- as well as of RAM-11-immunoreactive macrophages. We conclude that apoptotic macrophages located in atherosclerotic vessel wall are activated, antigen-presenting, integrin-expressing and oxidatively stressed cells. Since all these processes have been demonstrated to cause apoptosis of macrophages in vitro, we propose their potency accelerates the susceptibility of the macrophages to programmed cell death in atherosclerotic lesions.     

年龄对颈动脉形态特征及颈动脉斑块的影响

目的探讨年龄对颈动脉形态特征与颈动脉斑块形成的影响,明确老年患者颈动脉超声筛查对心血管事件的意义。方法连续选择在解放军总医院健康体检者976例,其中男性461例,女性515例,年龄29~86(58.8±13.8)岁。根据人群年龄分为青年组151例、中年组317例和老年组508例,对3组人群进行颈动脉超声检查,并对3组人群的颈动脉舒张末内径、内膜中层厚度、血液峰流速、搏动指数、阻力指数及斑块信息进行统计分析。结果3组人群颈动脉斑块在颈总动脉、窦部、颈内动脉、颈外动脉分布率分别为16.9%、58.5%、18.9%、5.7%,在窦部的分布率最高。3组人群颈动脉斑块随着年龄增长,斑块检出率显著升高(20.52%vs 35.65%vs 39.76%,P=0.000)3组人群随年龄的增长,颈内动脉和颈总动脉的血液峰流速及3条颈动脉的搏动指数均显著下降,青年组中年组老年组,差异有统计学意义(P0.01)。3条颈动脉的舒张末内径、内膜中层厚度、阻力指数和斑块检出率,还有颈外动脉的血液峰流速均显著升高,青年组中年组老年组,差异有统计学意义(P0.01)。结论年龄是导致颈动脉形态改变、颈动脉斑块形成与发展的重要因素,加强对老年患者的颈动脉超声筛查对心脑血管疾病的防治具有重要意义。