Aortas Of Cholesterol-fed Rabbits Research Articles

The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit: 1. Studies during the first two days after mild aortic injury

These studies examine the hypothesis that removal of aortic endothelium eliminates a barrier to lipoprotein cholesterol influx. The aortas of rabbits fed a cholesterol-rich diet from 7 to 16 days before balloon injury were studied 1 or 2 days after deendothelialization of specific areas of the aorta. By this design the aortic sterol content was near normal on the day of injury, but areas of injured and noninjured aorta were exposed to identical levels of elevated plasma cholesterol. Measuring the arterial [ 3H]- and [ 14C] cholesterol fractions accumulated during 2 different intervals after dosage in the same animals permitted calculation of total influx and fractional loss of aortic cholesterol. During the first 2 days after deendothelialization, total (unidirectional) cholesteryl ester influx in deendothelialized aorta was similar to that in adjacent uninjured aorta, but total influx of nonesterified cholesterol was increased. The calculated increase in influx of nonesterified cholesterol was not a result of an increase in hydrolysis of entered cholesteryl ester but probably represents increased exchange of labeled cholesterol between artery and plasma. These results suggest that subendothelial layers of the aorta of short-term cholesterol-fed rabbits function as significant barriers to lipoprotein influx and that processes other than increased permeation by lipoproteins initiate injury-induced lesions.

Comparison of lipid accumulation and metabolism in carrageenan-induced granulomas to aorta and blood monocytes of normal and cholesterol-fed rabbits

New Zealand rabbits (six each) were either maintained on a standard chow diet (ND) or the chow diet supplemented with cholesterol/peanut oil (HD) for 2 weeks. After 2 weeks, each animal had 15 ml of a 1% carrageenan gel injeccted subcutaneously into the midabdominal area. Rabbits were maintained on the diets for an additional 4 weeks. At sacrifice, blood was collected both for serum and for monocyte isolation and granulomas and aorta were excised. Tissues were assayed for lipid composition and lipid metabolism. Electron and light microscopies were performed on granuloma tissue. Granulomas from ND animals did not stain with oil red O. Granulomas from HD animals had homogenous oil red O staining indicating lipid accumulation. Granulomas from both ND and HD animals consisted of macrophages. Macrophages from ND rabbits accumulated follicular carrageenan but not lipid, while HD macrophages had the appearance of foam cells. Granuloma lipid content and metabolism closely paralleled the aorta and blood monocytes. The HD tissue had increased acylCoA:cholesterol acetyltransferase (ACAT) activity and lipid composition changes reflective of the atherosclerotic process. ND granulomas had no elevation of lipid content or ACAT. The carrageenan-induced granulomas provide a useful model for studying the biochemical and morphologic changes characteristic of arteries undergoing atherogenic change.

Potentiation of atherosclerotic lesions in rabbits by a high dietary level of vitamin E.

1. Two experiments were conducted to determine whether or not high dietary levels of vitamin E affect the development of atherosclerotic lesions in aortas of cholesterol-fed (5 g/kg diet) rabbits that were mechanically deendothelialized by balloon catheterization. 2. In the first experiment, the aortas of rabbits fed 2000 mg vitamin E/kg diet (i.e. 50-fold their nutritional requirement) for 8 weeks showed no gross morphological differences, either within or outside experimentally damaged areas, from those of rabbits fed the nutritionally adequate control level (40 mg/kg) of the vitamin. 3. In the second experiment, rabbits fed 10,000 mg vitamin E/kg diet (i.e. 250-fold requirement) for 14-15 weeks showed significantly greater endothelial loss and plaque formation at aortic sites outside of the mechanically damaged area than did controls. Plasma cholesterol levels were very high (9000-14,000 mg/l) and were not affected by dietary vitamin E level until 10-12 weeks when they were reduced moderately (18%). 4. It is concluded that very high levels of vitamin E can potentiate spontaneous atherosclerotic lesions, and it is suggested that this effect may depend on high cholesterol status.

Effect of hyercholesterolemia on cholinephosphotransferase activity in rabbit and rat vessel walls

The activity of cholinephosphotransferase (EC 2.7.8.2, CPT) which catalyses de novo synthesis of phosphatidylcholine (PC) was studied in aortas of rabbits and rats, and in brain microvessels of rabbits with a cholesterol feeding-induced hypercholesterolemia. Cholesterol feeding produced a marked atheromatous change in rabbit aortas but not in rat aortas. The aortas of cholesterol-fed rabbits displayed a significantly higher CPT activity than the controls. On the other hand, the aortic CPT activity of cholesterol-fed rats was not different from that of control rats. The brain microvessels of cholesterol-fed rabbits having atheromatous aortic lesions did not show any lipid deposition, and CPT activity was similar to that of control rabbits. A tocopherol-deficient, high-cholesterol diet produced microscopical lipid deposits in rat aortas, and CPT activity of these aortas was significantly higher than that of aortas of rats on tocopherol-supplemented diets containing either a normal or high amount of cholesterol. The increase in CPT activity in atheromatous lesion might be closely related to lipid deposition in vessel walls and may be a cause of the increase in PC content in these lesions. Further studies are required to clarify the mechanism of activation of CPT activity in atheromatous conditions.

Antiatherogenic properties of calcium channel blockers

Human atherosclerotic disease is characterized by focal accumulation of lipids, fibrous matrix materials, and calcium over several decades of life. A major difficulty in determining the efficacy of antiatherogenic agents has been the lack of appropriate animal models that reproducibly mimic the human arterial lesion. Studies in cholesterol-fed rabbits have become the “universal” model for testing therapeutic agents. Despite the difficulties in obtaining reproducible results with this animal model system, it is well-documented that high doses of calcium channel blockers can significantly reduce the accumulation of cholesterol and lipid in the aortas of cholesterol-fed rabbits. Isradipine is the only calcium channel blocker that has been shown to inhibit arterial cholesterol accumulation in the rabbit model at doses that approximate the human dose range. A dietary modification of the standard cholesterol-feeding regimen may provide an improved animal model for studies in atherogenic lesion proliferation. Such animal model experiments have provided the basis for the first prospective trials on the progression of human coronary artery disease using long-term administration of calcium channel blockers.

コレステロール食負荷ウサギにおける動脈壁脂質蓄積

コレステロール食負荷ウサギにおける動脈壁脂質蓄積

Effect of verapamil on accumulation of free and esterified cholesterol in the thoracic aorta of cholesterol-fed rabbits.

In order to investigate the effect of the calcium antagonist verapamil on atherogenesis in cholesterol-fed rabbits, 3 groups of 11 animals were fed a 2% cholesterol-enriched diet for 10 weeks. One group received verapamil in a daily dose of 16 mg/kg orally plus 2 mg/kg subcutaneously. This dosage resulted in plasma concentrations of verapamil in the same range as the usual therapeutic levels in humans. Another group received verapamil in a daily dose of 8 mg/kg orally and 0.5 mg/kg subcutaneously. The third group received placebo capsules orally and isotonic saline subcutaneously. Total cholesterol concentrations in plasma over the 10 weeks were 37 +/- 4, 42 +/- 4 and 45 +/- 3 mM (mean +/- SE) in the high verapamil-, in the low verapamil- and in the placebo group, respectively. These values were not significantly different. The distribution of cholesterol between HDL, LDL and VLDL in plasma was similar in the 3 groups. The high verapamil group had a significantly (P less than 0.05) lower concentration of cholesterol in the thoracic aorta than the placebo group (29 +/- 5 vs 43 +/- 9 mumol/g wet weight). The low verapamil group and the placebo group had the same aortic cholesterol concentrations. Neither dosage of verapamil affected the permeability of the aortic endothelium to plasma lipoproteins and albumin, as measured by use of radioactive tracers at the end of the experiment.

Effect of Probucol, Pantethine and Their Combinations on Serum Lipoprotein Metabolism and on the Incidence of Atheromatous Lesions in the Rabbit

AbstractEffect of probucol. pantethine and their combinations on serum lipoprotein metabolism and on the incidence of atheromatous lesions in aorta and coronary artery was studied in cholesterol-fed rabbits. Probucol ireatment (0.5% in diet) resulted in reducing HDL cholesterol and serum apo A-l levels significantly, while paniethine treatment (0.25%–0.75% in diet) tended to increase HDL cholesterol and serum apo A-l levels. Combined treatment with these two drugs showed a significant prevention in the reduction of HDL cholesterol and serum apo A-l levels by probucol alone. Probucol or pantethine treatment reduced effectively (V)LDL cholesterol and serum apo B levels, and these effects were accelerated additively when the two drugs were given concurrently. Atheromatous lesions in aorta and coronary artery in cholesterol-fed rabbits were prevented by the treatment with probucol (0.5% in diet) or pantethine (0.75% in diet) for 24 weeks. The combined treatment with these two drugs showed more marked prevention than either drug alone. From these findings, it is concluded that the combined treatment of probucol with pantethine is effective for improvement of serum lipoprotein disorders and for prevention of the incidence of atheromatous lesions in aorta and coronary artery in cholesterol-fed rabbits.

高脂血症ウサギのリポ蛋白代謝に及ぼすパンテチンとプロブコールの併用効果

Combined effect of pantethine and probucol on serum lipoprotein metabolism and on the incidence of atheromatous lesions in aorta and coronary artery was studied in cholesterol-fed rabbits.Probucol treatment (0.5% in diet) resulted in reducing HDL-cholesterol and serum apo A-I levels significantly, while pantethine treatment (0.25-0.75% in diet) tended to increase HDL-cholesterol and serum apo A-I levels. Combined treatment with two drugs showed a significant prevention in the reduction of HDL-cholesterol and serum apo A-I levels by probucol alone. Probucol or pantethine treatment reduced (V) LDL-cholesterol and serum apo B levels effectively and these effects were accelerated additively when the two drugs were given concurrently.Atheromatous lesions in aorta and coronary artery in cholesterol-fed rabbits were prevented by probucol (0.5% in diet) or pantethine (0.75% in diet) for 24 weeks. The combined treatment with two drugs showed more marked prevention than either drug alone.From these findings, it is concluded that the combined treatment of probucol with pantethine is effective for improvement of serum lipoprotein disorders and for prevention of the incidence of atheromatous lesions in aorta and coronary artery in cholesterol-fed rabbits.

Radioiodinated Cholesteryl lopanoate as a Potential Probe for the in Vivo Visualization of Atherosclerotic Lesions in Animals.

Radioiodinated cholesteryl iopanoate, a nonhydrolyzable cholesteryl ester probe, showed increased uptake into atherosclerotic aortas of cholesterol-fed rabbits in comparison with normal rabbits. Auto-radiography of the aortas showed the radioactivity to be concentrated in areas of visible atherosclerotic involvement. Lipid extraction and thin-layer chromatography of this tissue as well as liver, adrenal, and plasma confirmed the resistance of this probe to hydrolysis. These findings suggest that (125)I-cholesteryl iopanoate may prove useful for noninvasively monitoring atherosclerosis in intact laboratory animals.

Influx of [ 3H, 14C]cholesterol-labelled lipoprotein into re-endothelialised and de-endothelialised areas of ballooned aortas in normal-fed and cholesterol-fed rabbits

The entry of [ 3H]- and [ 3H, 14C]cholesterol-labelled lipoprotein into de-endothelialised and re-endothelialised areas of balloon-injured rabbit aortas was studied in normal-fed and cholesterol-fed rabbits. Studies were carried out 11–15 weeks after the initial injury when endothelial regeneration involved approximately half of the aortic area. The entry into the aorta of 3H-labelled free and ester cholesterol in lipoprotein over a 72-h period was studied following the ingestion of a single dose of 3H-labelled cholesterol. The entry of double labelled [ 3H, 14C]cholesterol-labelled lipoprotein was also studied over a 6-h period following the injection of plasma from dinor rabbits. The accumulation of cholesterol and cholesterol ester in the aorta in both the normal- and cholesterol-fed rabbits was significantly greater for the re-endothelialised (white) areas than for the de-endothelialised (blue) areas or the sham-operated aortas. Where the rabbits were cholesterol-fed 4–10 times the amount of cholesterol accumulated in re-endothelialized intima compared to normal intima. Both entry (μg/day/100 mg wet weight aortic intima) and clearance (μl plasma/ day/cm 2) of free and ester cholesterol were increased in the neointima compared with the normal intima for both normal-fed and cholesterol-fed rabbits. Hydrolysis of cholesterol ester occurred in the neointima and was greater than in the corresponding de-endothelialised area but less than for the sham-operated intima. Synthesis of cholesterol ester was minimal in all areas. Removal of labelled cholesterol and cholesterol ester from the intima during a 20-h efflux period following the initial 72-h loading period indicated that for aortas of both normal-fed and cholesterol-fed rabbits, there was greater removal for normal intima than for either re-endothelialised or de-endothelialised intima. However, no clear difference between the blue and white areas was observed. It is concluded that the accumulation of cholesterol in neointima after balloon injury is associated with a marked increase in permeability to lipoprotein of the neointima as well as to possible binding of lipoprotein to glycosaminoglycan in the artery.

Identification of potential antiatherosclerotic/hypolipidemic agents by their effect on hepatic conversion of androst-4-ene-3,17-dione to etiocholanolone and androsterone

Clinical observations have shown that hypercholesterolemia is associated with abnormal androgen metabolism, viz. an increased excretion of etiocholanolone (E) relative to androsterone (A). Substances which restore the A E ratio to normal likewise lower serum cholesterol. Postulating that the abnormal steroid and sterol metabolism may be either causally related or dependent on the same metabolic defect, we have developed in vitro and in vivo models to select drugs which favorably effect the ratio of A to E produced from [4- 14C]androst-4-ene-3,17-dione [4- 14C]A-dione). The in vitro model employs a mixture of rat liver microsomal Δ 4-3-ketosteroid-5 α-reductase and cytosolic 3a-hydroxysteroid dehydrogenase and Δ 4-3-ketosteroid-5 α-reductase. Kinetic and mechanistic studies have been performed on active compounds using this in vitro assay. The in vivo model employs i.v. injection of [4- 14C]A-dione followed by collection of bile in anesthetized, hypophysectomized female rats. Many compounds preselected in the in vitro assay likewise reduced the A E ratio in vivo. One of these compounds (CGS 10614A) also lowered serum cholesterol and reduced the incidence and severity of atherosclerotic lesions in aortas of cholesterol-fed rabbits.

高コレステロール食負荷家兎の大動脈脂質含有量に及ぼすMelinamide(アルテス)の影響

高コレステロール食負荷家兎の大動脈脂質含有量に及ぼすMelinamide(アルテス)の影響

The effect of bezafibrate and clofibrate on microsomal ACAT and lysosomal cholesterol ester hydrolase activity in the cholesterol-fed rabbit aorta

Bezafibrate markedly reduced the activity of fatty acyl CoA:cholesterol acyltransferase (ACAT) in the microsomal fraction of aortas from cholesterol-fed rabbits, while clofibrate was a less potent inhibitor. The activity of lysosomal cholesterol ester hydrolase (LCEH) was not significantly affected by either agent, indicating that inhibition of ACAT rather than stimulation of LCEH is a mechanism whereby these agents may decrease the cholesterol ester content of atherosclerotic aorta.

Transfer of plasma lipoprotein components and of plasma proteins into aortas of cholesterol-fed rabbits. Molecular size as a determinant of plasma lipoprotein influx.

The arterial influx of esterified and free cholesterol from low density lipoproteins and very low density lipoproteins in 20 hypercholesterolemic rabbits was measured simultaneously by the use of lipoproteins labeled in vivo with [3H]- and [14C]-cholesterol. The simultaneous arterial influx of either [3H]-leucine-labeled very low density lipoproteins, low density lipoproteins, high density lipoproteins, or plasma proteins was also measured in each rabbit. The arterial influx was calculated as intimal clearance, i.e., the influx of a given fraction divided by its plasma concentration. The intimal clearance of low density lipoprotein esterified cholesterol was equal to that for the apolipoproteins of that fraction, which is compatible with an arterial influx of intact low density lipoprotein molecules. The intimal clearance of very low density apolipoprotein or cholesteryl ester was less than that for low density lipoprotein, whereas high density lipoprotein and albumin clearances exceeded low density lipoprotein clearance by 1.5- to 3-fold. The intimal clearances of plasma proteins, high density, low density, and very low density lipoproteins decreased linearly with the logarithm of the macromolecular diameter. This indicates that the arterial influx of three plasma lipoprotein fractions and of plasma proteins proceeds by similar mechanisms. Apparently the relative intimal clearances of lipoproteins are more dependent on their size relative to pores or vesicular diameters at the plasma-artery interface than on specific interactions between lipoproteins and the arterial intimal surface.

Effect of cholesterol feeding on arterial lipolytic activity in the rabbit

Two trioleoyl glycerol hydrolases, one of lysosomal origin as determined by a high correlation with the lysosomal marker enzyme, N-acetyl-β-glucosaminidase, and one having the characteristics of lipoprotein lipase, were measured at varying stages of lesion development in the aortas of cholesterol-fed rabbits. Both lipases were greatly enhanced in atheromatous aortas and were linearly related to lesion severity as measured by total aortic cholesterol. Lipoprotein lipase activities of myocardium and of plasma of cholesterol-fed rabbits were also significantly increased relative to controls. The data suggest that lipoprotein lipase might be a factor regulating cholesterol deposition in the aorta.

Hypertension-accelerated atherogenesis in cholesterol-fed rabbits

Entry of 125I-labelled low density lipoprotein ([ 125I]LDL) into the aortic intima was studied over 6 hours in normotensive and hypertensive rabbits fed a 1% cholesterol diet for 9 and 4 weeks respectively. Studies were also made in hypertensive and normotensive cholesterol-fed rabbits in which blood pressure was reduced acutely with parenteral hydralazine. In all groups the entry of [ 125I]LDL was greatest in the aortic arch and significantly less in both the descending thoracic and abdominal regions. Lipoprotein entry into the aorta of cholesterol-fed rabbits was increased some 10-fold over the corresponding value previously found in rabbits fed a normal diet [1]. This increase was due to increased vascular permeability as well as to increased plasma LDL concentration. The hypertensive cholesterol-fed rabbits did not show significantly greater entry of [ 125I] LDL than the normotensive cholesterol-fed rabbits. Comparison of the rate of LDL entry over 6 hours and the quantity of cholesterol accumulated in the aortic segments over the period of cholesterol feeding indicated that lipoprotein fractions other than LDL must contribute significant amounts of cholesterol to the developing lesion. The finding that LDL entry paralleled accumulation during cholesterol feeding, together with the finding that acute reversal of hypertension did not reduce the entry of [ 125I]LDL suggest that mechanisms other than increased filtration of plasma low density lipoprotein contribute significantly to the accelerated development of atherosclerosis in hypertension.

Uptake and metabolism of 3H-fatty acid labelled lecithin by normal and atherosclerotic intima in vivo and in vitro

Following the intravenous injection of tracer doses of 3H-fatty acid labelled lecithin into normally fed rabbits, minimal incorporation into serum cholesterol ester over a 6-hour period was observed. The labelled phospholipid was rapidly removed from the serum primarily by R.E. tissues with little uptake by the aorta. The uptake and metabolism of 3H-fatty acid labelled lecithin by explants of normal and cholesterol-fed rabbit aortas was also investigated in vitro for periods up to 8 days. Low uptake and incorporation into cholesterol ester was observed for the normal aortic explants. The labelled phospholipid was taken up to a greater extent by explants from cholesterol-fed rabbit aortas and the fatty acid incorporated into both triglyceride and cholesterol ester in the explants. The incorporation of fatty acid into cholesterol ester from phospholipid was associated with hydrolysis of the phospholipid and re-incorporation of the fatty acid into cholesterol ester, rather than by the action of lecithin cholesterol acyl transferase.

Effect of hypertension on the in vitro incorporation of [1-14C]-oleate into phospholipid and cholesterol ester in the aortae of normal-fed and cholesterol-fed rabbits.

The effect of prior hypertension on the in vitro incorporation of [1-14C]-oleate into phospholipid and cholesterol ester in aortae from cholesterol-fed and normal fed rabbits was studied. Incorporation of [1-14C]-oleate into phospholipid was not increased in aortae from either hypertensive normal-fed or hypertensive cholesterol-fed rabbits when compared to the appropriate normotensive controls. In the normal-fed rabbits, incorporation of [1-14C]-oleate into cholesterol ester was increased by hypertension in all aortic regions. In cholesterol-fed rabbits cholesterol esterification was found to be proportional to the intimal cholesterol concentration, irrespective of the prior blood pressure or the particular aortic region studied. It is concluded that the increased lipid synthesis in atherosclerotic vessels from hypertensive rabbits is a consequence of the increased lipid accumulation produced by hypertension and not the result of hypertension directly stimulating arterial wall metabolism.

Incorporation in vitro of 14C-labelled acetate and 32P-labelled phosphate into lipid in thoracic aortae from hypertensive and nomotensive rabbits

The effect of prior hypertension on lipid synthesis in the thoracic aortae of normal-fed and cholesterol-fed rabbits was studied in vitro using [1− 14C]acetate and [ 32P]phosphate as lipid precursors. In normally fed rabbits, prior hypertension did not increase the incorporation of the labelled precursors into either phospholipid or neutral lipid. In cholesterol-fed rabbits, hypertension increased the incorporation of [ 32P]phosphate into phosphatidyl-choline and of [1− 14C]acetate into cholesterol ester. The increased incorporation of [1− 14C]acetate into cholesterol ester was accompanied by an increase in intimal total cholesterol concentration. For both normotensive and hypertensive cholesterol-fed rabbits there was a close correlation between cholesterol esterification and total cholesterol concentration of the thoracic intima. It is concluded that the increase in aortic lipid synthesis in hypertensive cholesterol-fed rabbits is secondary to the increased cholesterol accumulation induced by hypertension rather than to a direct stimulation of arterial wall lipid synthesis by hypertension per se.