Ganglioside content and composition of cells from normal and atherosclerotic human aorta

The ganglioside content and composition of cells obtained by enzyme digestion of 2 layers of human aortic intima were investigated. Five gangliosides were identified in cells isolated from the external musculo-elastic intimal layer adjacent to the media: GM3, GM1, GD3, GD1a, and GT1b. The same gangliosides plus ganglioside Gx, the chromatographic mobility of which corresponded to the mobility of ganglioside GD1b from human brain, were found in cells from the internal elastic-hyperplastic intimal layer adjacent to the vessel lumen. In both layers, the major cellular ganglioside was GM3 which represented 60% of the total cellular ganglioside content. The ganglioside content was lower in cells obtained from fatty streaks compared to cells isolated from unaffected intima. The amount of di- and trisialogangliosides in atherosclerotic plaque cells was lower, and that of monosialogangliosides higher than in cells isolated from unaffected intima. The amount of GM3 was mainly responsible for the difference in the total ganglioside content of cells obtained from different lesion types. On the whole, cells from fatty streaks contained smaller amounts of total gangliosides, whereas cells from plaques had greater total ganglioside content, than cells from unaffected intima.     

Neutral glycosphingolipid content and composition of cells from normal and atherosclerotic human aorta.

We have investigated the content and composition of neutral glycosphingolipids (GSLs) in the cells isolated by enzyme digestion from elastic-hyperplastic and musculo-elastic intimal layers of grossly normal and atherosclerotic regions of human aorta. We have detected three types of neutral GSLs in the intimal cells identified as glucosylceramide, trihexosylceramide and tetrahexosylceramide. We failed to detect lactosylceramide in the intimal cells. The cells of the elastic-hyperplastic layer of grossly normal regions contained trihexosylceramide and tetrahexosylceramide, while glucosylceramide was not detected. Considerable amounts of glucosylceramide were found, and the trihexosylceramide and tetrahexosylceramide content was increased in the cells isolated from atherosclerotic regions. The cells of the musculo-elastic layer of grossly normal intimal regions contained glucosylceramide, trihexosylceramide and tetrahexosylceramide. Cells of the musculo-elastic layer of the fatty streak contained noticeable higher amounts of glucosylceramide, as well as greater amounts of trihexosylceramide and tetrahexosylceramide. Cells of the musculo-elastic layer of the plaque also appeared to contain more glucosylceramide, tetrahexosylceramide, but less trihexosylceramide as compared with grossly normal regions. In both cases cells of the fatty streak exhibited the highest total amount of neutral GSLs, but at the same time the neutral GSL composition of the fatty streak was not similar to GSL composition which is known for human blood monocytes. These findings indicate that elevation of neutral GSL level is observed in cells from atherosclerotic lesions of human aortic intima.     

Atherogenic properties of beta adrenergic blockaders evident on vascular wall cells

In cultured human cells, beta-adrenoblockers such as propranolol, alprenolol, metoprolol, atenolol, pindolol, and thymolol, as well as sera from patients with coronary heart disease were examined for atherogenic activity following a single administration of propranolol (80 mg) and pindolol (10 mg). Addition of the beta-adrenoblockers to the cell culture was demonstrated to enhance cellular total cholesterol levels and to stimulation their proliferation. Propranolol and pindolol given in a single dose was found to result in the appearance of atherogenic properties of the patients' sera.     

Localization of apolipoprotein E in normal and atherosclerotic human aorta

To elucidate the role of apolipoprotein E (apo E) in atherogenesis, we have investigated the localization of apo E in normal and atherosclerotic aortas as well as in other tissues of 32 post-mortem individuals. Using double immunofluorescence it has been found that normal intima of individuals older than 20 years and some adolescents contained immunoreactive material that reacted with poly- and monoclonal antibodies to apo E. A staining pattern of apo E differed from that of apolipoprotein B, the latter being seen in normal intima of each child older than 7 years. Apo E was present extracellularly in lipid streaks and atheromatous plaques, where its staining was particularly intensive around the necrotic zone of plaques. Some macrophages in the plaques of 4 aortas exhibited apo E-positive staining, while aortic endothelial and smooth muscle cells never contained apo E. Apo E-positive staining was not found in the majority of vessel cells, it was always, however, observed in other types of cells including hepatocytes. Kupffer cells, spleen macrophages and cerebral astrocytes. Our findings indicate that only some macrophages in human aorta may be responsible for the production of apo E that can participate in reverse cholesterol transport. At the same time, apo E accumulation in the aortic wall may promote the development of atherosclerosis.     

Agents that increase cellular cyclic AMP inhibit proliferative activity and decrease lipid content in cells cultured from atherosclerotic human aorta

Cholera toxin, methylisobutylxanthine and prostacyclin (PGI2) analogues as well as dibutyryl cyclic AMP inhibit by 2-7-fold 3H-thymidine uptake into intimal cells isolated from atherosclerotic human aorta in primary culture. These agents also decrease cholesteryl ester and triglyceride levels and do not affect content of phospholipids and free cholesterol in cells cultured from atherosclerotic lesions.     

Growth properties and composition of cytoskeletal and cytocontractile proteins in aortic cells isolated and cultured from normal and atherosclerotic rabbits

Aortic intima-medias of normal and cholesterol-fed rabbits were studied with EM and cells were isolated by enzyme digestion. The composition of cytoskeletal and cytocontractile proteins was determined with SDS-PAGE and the primary growth and thymidine incorporation rates were assessed after seeding the cells into tissue culture flasks. Ultrastructurally, the SMCs in the thickened atherosclerotic intima differed from the contractile medial SMCs in containing lipid vacuoles, enlarged endoplasmic reticulum and a reduced number of myofilaments, thus showing characteristics of dedifferentiated SMCs. In SDS-PAGE, freshly isolated cells from the atherosclerotic intima-medias had a lower content of myosin and actin, and a higher proportion of vimentin and desmin than SMCs from normal aortas. Enzyme-isolated SMCs from normal aortas did not start to grow and incorporate radioactive thymidine until 5-6 days after seeding, whereas those from atherosclerotic aortas did so within 2 days. After a week in culture, SMCs from both sources resembled each other, and had decreased contents of myosin and actin, and increased concentrations of vimentin in comparison to freshly isolated normal SMCs. The present results indicate (a) that morphological dedifferentiation of SMCs in aortic lesions of cholesterol-fed rabbits is associated with an increased proportion of the proteins of the intermediate filaments and a decrease in those of the thin and thick myofilaments as determined with SDS-PAGE, and (b) that similar changes take place when normal SMCs are cultured in vitro. The results also suggest (c) that enzyme-isolated atherosclerotic SMCs proliferate in a primary culture without the lag period that normal SMCs apparently require for dedifferentiation.     

Electrophysiologic effects of beta blockers in ventricular arrhythmias

Beta-adrenergic receptor blocking agents are effective antiarrhythmic drugs in patients with ventricular arrhythmias. However, these agents exert little or no measurable electrophysiologic effect on normal Purkinje and ventricular muscle fibers when administered acutely. They prevent catecholamine-induced increases in Purkinje fiber automaticity and may interfere with catecholamine-dependent slow responses. beta-adrenergic blocking drugs also prevent the decrease in ventricular fibrillation threshold induced by catecholamines. In the acutely ischemic ventricle, some beta blockers selectively depress conduction within the ischemic zone. The long-term administration of some beta blockers has, in contrast to their short-term effects, been shown to prolong action potential duration and effective refractory period in the ventricle. Which of these observed electrophysiologic effects, either alone or in combination, contributes to the ventricular antiarrhythmic effects of beta-blocking drugs in man is at present unknown.     

Apolipoprotein B retention in the grossly normal and atherosclerotic human aorta.

Apoliporotein B (apoB) was measured in buffer-extracted homogenates of grossly normal and artherosclerotic human aortic intima by means of an electroimmunoassay procedure. The apoB values which were expressed as microgram per mg tissue dry weight, varied widely, ranging from 0.34 to 18.45 in normal intima and from 0.8 to 12.5 in fatty fibrous plaques. No consistent differences in apoB content were found between normal intimas from thoracic and abdominal aortic regions. There was a statistically significant positive correlation between the quantity of buffer-extractable apoB in normal regions and the plasma cholesterol and triglyceride concentration. Buffer-extractable apoB values were significantly higher in fatty fibrous plaques than in ulcerated lesions from the same vessel. However, fatty fibrous plaque apoB values were significantly lower than those from grossly normal regions from the same aorta, although the topographical distribution of apoB was more widespread in plaques than in normal regions, as shown by immunofluorescence studies. This apparent discrepancy reflected the incomplete extraction of apoB from plaques as contrasted to normal regions. The relatively loosely bound apoB, extractable by standard buffers, may represent intact low density lipoprotein (LDL) and/or very low density lipoprotein (VLDL), while the tightly bound fraction may represent insoluble complexes of intact lipoproteins within the plaque or delipidated apoB.     

Stable analogues of prostacyclin and thromboxane A2 display contradictory influences on atherosclerotic properties of cells cultured from human aorta. The effect of calcium antagonists

We examined the influence of stable prostacyclin analogues (carbacyclin) and thromboxane A2 (U46619) on atherosclerotic properties of cells: [3H]thymidine incorporation and intracellular cholesterol content. A primary culture of human aortic subendothelial cells derived from atherosclerotic plaques was used. Carbacyclin exerted a direct anti-atherosclerotic effect, significantly reducing atherosclerotic manifestations of cells, while agent U46619 stimulated proliferation and cholesterol accumulation, i.e. demonstrated atherogenic potency in culture. Calcium antagonists (verapamil and diltiazem) markedly enhanced anti-atherosclerotic properties of carbacyclin and restricted the atherogenicity of U46619.     

Impact of beta 1 selectivity and intrinsic sympathomimetic activity on potential unwanted noncardiovascular effects of beta blockers

Beta-adrenoceptor-blocking drugs are widely used as effective antihypertensive and antianginal agents, but treatment with these agents may be contraindicated in hypertensive patients in whom receptor blockade would interfere with noncardiovascular activities dependent on sympathetic drive. beta blockade impairs pulmonary function in asthmatic patients through antagonism of beta 2 bronchodilation. However, patients with chest problems may be treated effectively with beta 1-selective drugs, including acebutolol, atenolol and metoprolol. The metabolic response to hypoglycemia, which is mediated by beta-receptor stimulation, involves insulin release, gluconeogenesis, tachycardia and increased systolic pressure. Beta 1-selective drugs are preferred in patients who need to increase blood glucose levels because they do not interfere with glycogenolysis. Hypertension induced by pregnancy may be treated with a beta blocker with no apparent adverse effects on the fetus or neonate. Those possessing intrinsic sympathomimetic activity may be preferable.     

Collagen-synthesizing cells in initial and advanced atherosclerotic lesions of human aorta

Accumulation of extracellular matrix, fibrosis, is regarded to be one of the major manifestations of atherosclerosis. Collagen type I is the predominant matrix component in human atherosclerotic plaques. In this work we have demonstrated procollagen type I expressing cells (PCl-cells) and studied their localization in grossly normal human aorta and atherosclerotic lesions: initial lesions, fatty streaks, fibrolipid lesions (fibrolipid plaque, fibroatheroma), fibrotic lesions (fibrous plaque). PCl-cells were revealed immunocytochemically using SPI.D8 monoclonal antibody against human procollagen type I. We failed to detect PCl-cells in the areas of grossly normal aorta and media underlying atherosclerotic lesions. Positively stained cells were shown in the areas of initial lesions, fatty streaks, fibrolipid and fibrous plaques. The largest amount of PCl-cells was revealed in fatty streaks. These cells were predominantly localized in the preluminal proteoglycan-rich intimal sublayer. Intimal cells in grossly normal regions formed a common cellular network contacting each other with their processes. The cellular network is found to be partly disintegrated in atherosclerotic lesions, which leads to the appearance of isolated cells. The share of isolated PCl-cells localized outside the intimal cellular network was higher in advanced lesions than in the areas of early atherosclerotic lesions. In initial lesions most of PCl-cells were identified as smooth muscle cells using antibodies to smooth muscle -actin. In fatty streaks PCl-expressing smooth muscle cells were fewer in number. Much fewer cells double-stained with anti--actin and anti-PCI antibodies were found in fibrolipid and fibrous plaques. The proportion of these double stained cells was higher among total number of PCl-cells involved in the cellular network versus PCl-cells outside the network. The results of the study demonstrated that the most active de novo synthesis of interstitial collagen takes place in the regions of atherosclerotic lesions characterized by lipid deposition, which may lead to the further progression of atherosclerotic lesions.     

Hematoporphyrin binding to cultured aortic smooth muscle cells from spontaneously atherosclerotic turkey

Porphyrins are known to be accumulated in vivo by tumors and atherosclerotic plaques. We studied the interaction of cultured aortic smooth muscle cells (SMC) from spontaneously atherosclerotic Broad Breasted White Turkeys (BBWT) with free hematoporphyrin (Hp) and low density lipoprotein (LDL)-Hp complexes. A significantly higher binding of LDL-Hp to SMC as compared to free Hp was observed. These data indicate that porphyrin binding to vascular SMC represents a possible mechanism for porphyrin accumulation by atherosclerotic plaques. This process is mediated, at least in part, by LDL.     

Histological and angiographic effects of a pulsed holmium:YAG laser in normal and atherosclerotic human coronary arteries and aorta.

OBJECTIVE: The aims were (1) To determine the histological and angiographic effects of holmium:YAG laser energy delivered through clinical multifibre laser catheters on fresh cadaveric coronary arteries; and (2) to relate the placement of optical fibres in the catheter to patterns of tissue ablation in cadaveric aorta. METHODS: Eight fresh cadaveric hearts and segments of aorta were used. Hearts were mounted on a new pressure perfusion device. The laser catheter was delivered over a guidewire in the lumen until it met an area of resistance. The coronary artery lumen was perfused at approximately 100 mm Hg mean pressure. These arterial areas were identified on angiography, marked, and then exposed to laser energy in the range 600-3000 mJ.mm-2. Normal and atherosclerotic areas of fresh cadaveric aortic strips were exposed to increasing laser energies using either constant or increasing fluence. Coronary arteries were pressure perfused with formalin for 18-24 h at 100 mm Hg mean pressure, and aortic strips were immersed in 5% formalin. Light and scanning electron microscopy studies were carried out. RESULTS: There were no perforations or dissections by angiography in the fresh coronary arteries. One of 15 normal coronary artery segments and 10 of 16 of the pressure perfused, fixed, atherosclerotic coronary artery segments showed thermal changes associated with atherosclerotic plaque ablation. In aortic tissue, thermal effects extended 0 to 0.6 mm lateral to the ablated crater. Acoustic effects were seen only in the aortic strips after ablation at fluences > 1000 mJ.mm-2. The "dead spaces" around the optical fibres in the catheter resulted in significant amounts of coagulated tissue fragments remaining in the crater. CONCLUSIONS: Holmium:YAG laser energy delivered through multifibre catheters ablated atherosclerotic tissue in coronary arteries with minimal damage to the normal walls. The cadaveric coronary artery perfusion apparatus is useful for assessing catheter delivery and mobility and the effects of laser energy on the coaxially orientated normal and atherosclerotic coronary arterial wall.     

Differential effects of interleukin 1 alpha and 1 beta on cultured human and rat thyroid epithelial cells

The effects of human recombinant interleukin 1 alpha (20 pg/1-2 micrograms/l) and 1 beta (200 pg/1-20 micrograms/l) on two systems of thyroid cells have been compared. The thyroglobulin and cAMP secretion and the DNA content of human thyroid cells cultured in monolayer and of continuously grown rat thyroid cells, Fischer rat thyroid cell line have been studied. The growth of the rat thyroid cell line was inhibited by interleukin 1 beta (20 ng/1-20 micrograms/l), but not by interleukin 1 alpha. None of the cytokines changed the cAMP production of the rat thyroid cells. In contrast, both cAMP production and thyroglobulin secretion were inhibited dose-dependently by the cytokines in human thyroid cells in secondary cultures. These results caution the interpretation and extrapolation of changes induced by interleukin 1 from one cell system to the other.     

Use of cultured atherosclerotic cells for investigation of antiatherosclerotic effects of anipamil and other calcium antagonists.

Calcium antagonists have been shown to exhibit an antiatherosclerotic action in primary cultures of human aortic atherosclerotic cells by causing a reduction in intracellular lipid content, proliferative activity and synthesis of the extracellular matrix. Verapamil and nifedipine exhibited the highest efficacy in this respect. The new calcium antagonist, anipamil (racemate and enantiomers), has been tested in cell cultures. At 10(-6) M and higher concentrations, anipamil and its enantiomers produced considerable decrease in intracellular content of cholesteryl esters, triglycerides and free cholesterol, suppressed cell proliferation and inhibited synthesis of the extracellular matrix. The efficacy of anipamil (enantiomers and racemate) was similar to that of verapamil and greater than that of nifedipine. Plasma obtained from patients after administration of 80 mg verapamil or 20 mg nifedipine significantly lowered the cholesterol content of cultured cells. Blood plasma of most atherosclerotic patients possesses atherogenic properties, i.e., it is able to increase the cholesterol content in cultured cells. Plasma atherogenicity seen in cultures decreased considerably or even disappeared after both nifedipine and verapamil administration. After 28 days of nifedipine therapy, plasma atherogenicity was lower compared with the initial value at the beginning of the treatment. These observations suggest that control of plasma atherogenicity after drug administration may provide an additional tool for optimisation of direct antiatherogenic and antiatherosclerotic therapy.     

食鱼对年轻人主动脉壁的影响和动脉粥样硬化早期病变的研究

用国家“七五”课题方法，对５１例渔区（舟山、宁波）意外死亡年轻人（１５～３９岁）尸检主动脉动脉粥样硬化（ＡＳ）早期病变进行了研究。数据经统计学处理，并与国家“七五”课题资料结果相比较。显示：渔区人主动脉内膜嗜苏丹病变（ＳＬ）面积百分比明显小于南宁，更小于北京；胸主动脉壁蛋白聚糖（ＰＧ）总量渔区低于南宁（Ｐ＜０．０１），硫酸乙酰肝素－蛋白聚糖（ＨＳ－ＰＧ）相对百分含量略高于南宁（Ｐ＞０．０５），亦高于北京（Ｐ＜０．０５）；主动脉内膜平滑肌细胞核数密度渔区大于南宁（Ｐ＜０．０５），面密度小于北京（Ｐ＜０．０５），中膜平滑肌细胞核面密度明显小于南宁及北京（Ｐ＜０．０１）；渔区主动脉ＡＳ病变检出率：Ⅰ、Ⅱ级病变与南宁、北京无差异，Ⅲ、Ⅳ、Ⅴ级病变均明显轻于南宁和北京（Ｐ＜０．０１）。结果提示：多食海鱼的生活习惯是导致鱼区人ＡＳ群的病变程度轻的重要因素之一。     

In vitro effects of argon laser radiation the human healthy and atherosclerotic aorta

Argon laser exposures in vitro were done on human aortas. The laser energy applied on a fresh aorta section (A) was from 100mw to 1000mw. A second aorta section (B), formalin fixed, was irradiated under saline solution by a fiberoptic system. Laser energy was from 400mw to 720mw. The time exposures were all different in both sections. A and B histologic findings of thermal damage were similar Three zones of tissue injury were observed: I) crater because of tissue vaporization; II) coagulative necrosis surrounding it; and III) multiple vacuoles in the adjacent tissue produced by acoustic or shock injury. Tissue damage was related directly with total energy delivered: less than or equal to 500mw showed inner vascular wall necrosis, and at greater than or equal to 720mw it was perforated. Atherosclerotic tissue was more resistant to laser thermal injury than normal tissue. We could not find time relation with thermal damage. This preliminary information is an early step for the possible use of Argon laser on cardiovascular area.