Thomas E. Carew

Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.

Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, properties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was degraded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.

Initiation of atherosclerotic lesions in cholesterol-fed rabbits. II. Selective retention of LDL vs. selective increases in LDL permeability in susceptible sites of arteries.

We asked if the arterial sites most prone to early lesions in cholesterol-fed rabbits have higher permeabilities to low density lipoprotein (LDL) in normolipidemic rabbits or if these sites become more permeable shortly after the onset of cholesterol feeding. We also considered whether the focal increases in the concentration of LDL within the arterial wall in lesion-susceptible sites before fatty streak formation can be explained by increased arterial permeability to LDL or by other mechanisms such as decreased rates of LDL efflux or degradation. 125I-tyramine cellobiose-labeled LDL was injected 1 hour before death to determine the initial rate of LDL entry into lesion-prone and lesion-resistant sites of aorta as a measure of permeability. This was studied in normal rabbits and in rabbits fed cholesterol for 4, 8, or 16 days. Combining this permeability data with the tracer data described in the accompanying article, we fit a kinetic model to calculate the mass and mean residence time of intact LDL within the artery and the fractional rates of LDL degradation and efflux from the artery. In normal rabbits, the permeability of lesion-susceptible branch sites of the abdominal aorta was about four times that of the lesion-resistant, nonbranched areas. However, the permeability of the aortic arch, a susceptible site, was similar to that of the lesion-resistant descending thoracic aorta. Permeability to LDL did not increase in any aortic site during the 16 days of cholesterol feeding, even in sites with the largest increases in arterial LDL concentrations. Plasma LDL cholesterol concentration increased substantially and total LDL cholesterol delivery into the artery increased many fold. Since there was no differential change in permeability between susceptible and resistant sites, the increased entry of LDL did not explain the selective increases in arterial LDL concentration in susceptible sites. Kinetic analysis indicated that the fractional rate of degradation of the arterial LDL pool was lower in lesion-prone sites than in lesion-resistant sites in all animals. Fractional rates of efflux of arterial LDL decreased in lesion-susceptible branch sites of the abdominal aorta and were low in the lesion-susceptible aortic arch. These results suggest that the focal increases in LDL concentration observed in all lesion-susceptible sites of cholesterol-fed rabbits before fatty streak formation are due to localized differences in LDL retention and diminished fractional rates of LDL degradation, not to selectively increased permeability.(ABSTRACT TRUNCATED AT 400 WORDS)

Initiation of atherosclerotic lesions in cholesterol-fed rabbits. I. Focal increases in arterial LDL concentration precede development of fatty streak lesions.

We have reported that arterial low density lipoprotein (LDL) concentrations and degradation rates in normal rabbits are elevated in those aortic sites most susceptible to early atheromatous lesions resulting from cholesterol feeding. Here we asked whether the focal differences in LDL metabolism observed in normolipidemic rabbits are accentuated during the first 16 days of feeding 2% cholesterol and whether such changes occur before accumulation of significant numbers of macrophage foam cells. No lesions were grossly visible after 16 days of cholesterol feeding. Histology indicated that macrophage foam cells were sparse during this interval and were found only in lesion-prone sites at longer feeding times. We used LDL labeled both with 131I (to trace undegraded LDL), and with the intracellularly trapped ligand 125I-tyramine cellobiose (to trace degraded plus undegraded LDL). The most profound change was a marked and focal increase in the concentration of intact LDL within the arterial wall of lesion-prone sites. After 16 days of cholesterol feeding when the plasma LDL cholesterol concentration had increased 7.6-fold, the concentration of intact LDL in lesion-prone branch sites of the abdominal aorta was increased by 22-fold (from 2.5 to 54 micrograms LDL cholesterol/g). These concentrations were two and 5.7 times, respectively, as great as in the lesion-resistant, nonbranch areas of the abdominal aorta of the same animals. Similar, but less striking, results were found when the lesion-prone aortic arch and intercostal orifices of the thoracic aorta were compared with adjacent lesion-resistant sites. Arterial LDL degradation rates expressed in terms of LDL mass also increased with time of cholesterol feeding, and were greater in the aortic arch and in branch sites of the abdominal aorta than in adjacent lesion-resistant sites. However, fractional rates of degradation of arterial LDL were decreased in all sites by cholesterol feeding, and were lower in susceptible than in resistant sites. This was probably due in large part to saturation and down-regulation of LDL receptors. The timing, focal nature, and site-specificity of these changes suggest that an increased concentration of LDL in the arterial wall may be an early indication of lesion formation and, in fact, may be a necessary first step in the pathogenesis of the fatty streak lesion in cholesterol-fed rabbits.

Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman primate by probucol. I. Is the extent of atherosclerosis related to resistance of LDL to oxidation?

Lipoprotein oxidation is believed to play an important role in atherogenesis. To investigate whether inhibition of oxidation of low density lipoprotein (LDL) would alter atherogenesis in the nonhuman primate, we administered probucol, a potent antioxidant, to Macaca nemestrina fed a high-fat, high-cholesterol diet. Probucol was administered to half of the 16 monkeys 14 wk after starting the hypercholesterolemic diet, and was given daily until they were sacrificed after 11 mos. To evaluate the antioxidant effect of probucol, the resistance of isolated plasma LDL to in vitro oxidation was evaluated. Probucol significantly increased the resistance of LDL to oxidative modification, as shown by an increase in the lag time required for conjugated diene formation. Lesions in the probucol-treated animals appeared less mature, and increased accumulation of lipid was observed in smooth muscle cells. Comparison of all control and probucol-treated monkeys demonstrated that intimal lesion areas in the thoracic aortas of the probucol-treated monkeys were reduced by 43% (P < 0.0001), but no significant difference in lesion area was found in the abdominal aortas or in the iliac arteries. However, the lag phase of conjugated diene formation was not prolonged in 2 of the 8 probucol-treated animals. A plot of intimal lesion size versus lag phase of all 16 animals showed a trend that lesion size was inversely related to oxidation resistance for all anatomic sites. The strong inverse relationship between intimal lesion size and resistance of LDL to oxidation supports a role for lipoprotein oxidation in the development and progression of lesions of atherosclerosis. The possibility that some of the effect is due to other biological properties of probucol cannot be ruled out.

Regional differences in myocardial performance in the left ventricle of the dog.

To determine whether significant regional differences in shortening exist in the canine left ventricle, the shortening characteristics of small segments of the circumferentially oriented hoop axis fibers and the more longitudinally oriented fibers near the epicardium were examined using pairs of ultrasound crystals placed at three levels of the left ventricular free wall in the open-chest dog. Mean control shortening of the hoop axis fibers near the apex of the left ventricle averaged 20% of the end-diastolic length, significantly greater than shortening at the midventricular (13%) or basal (14%) levels. During transient periods of aortic constriction, end-diastolic length increased significantly and the extent of shortening was maintained for the hoop axis fibers at the apical and midventricular levels; end-diastolic length did not change and shortening decreased at the basal level. The epicardial fibers shortened an average of 5.6% of their end-diastolic length during control conditions at all three sites and showed small, parallel changes in shortening and end-diastolic length during aortic constriction. We conclude that significantly greater hoop axis shortening occurs near the apex of the left ventricle and that at this level a uniformly contracting model is inappropriate. In addition, the response of the hoop axis fibers to increased aortic impedance is not homogeneous, with a significant reduction in shortening occurring only at the base of the left ventricle where end-diastolic length does not increase.

Uptake and degradation of low density lipoprotein by swine arterial smooth muscle cells with inhibition of cholesterol biosynthesis

We have previously proposed on the basis of studies in hepatectomized animals that low density lipoproteins are degraded at a significant rate by peripheral tissues. To test the capacity of one peripheral cell type to catabolize low density lipoprotein, cultures of swine aortic smooth muscle cells were incubated with homologous 125I-labeled low density lipoprotein and uptake and degradation measured. Degradation of 125I-labeled low density lipoprotein to products soluble in trichloroacetic acid showed an initial lag period of 1--2 h after which the rate increased and remained linear for the following 15 h. Rates of degradation increased sharply with low density lipoprotein concentration over the lower range (from 0--25 mug protein/ml) and then more slowly up to the highest concentration tested, 300 mug protein/ml. Even at very low concentrations, 1 mug low density lipoprotein protein/ml (less than 10% of the plasma low density lipoprotein concentration), the in vitro degradation rate (per kg of smooth muscle cells) exceeded the in vivo degradation rate (per kg of total body weight). To the extent that smooth muscle cells are representative of other peripheral cells, the results support the proposal that peripheral degradation of low density lipoprotein apoprotein may be quantitatively important. The rate of incorporation of labeled acetate into sterols was suppressed in cells incubated with whole serum, low density and very low density lipoproteins, or suspensions of free cholesterol. In this respect, the results were similar to those observed in human skin fibroblasts studied concurrently. However, high density lipoprotein inhibited sterol synthesis by about 25% in swine smooth muscle cells while it had no effect in human skin fibroblasts.

Oxidative modification of beta-very low density lipoprotein. Potential role in monocyte recruitment and foam cell formation.

Oxidative modification of low density lipoprotein (LDL) generates a form that Is degraded much more rapidly by macrophages and may thus be more atherogenic than unoxldized LDL. Recently, we provided evidence that oxidative modification of LDL may play a significant role In the generation of fatty streaks in the LDL receptor-deficient rabbit The major lipoprotein in cholesterol-fed animals is the 0-very low density lipoprotein (β-VLDL). Since β-VLDL Is avidly taken up by macrophages, It could lead to foam cell formation without the need for oxidative modification or modification of other kinds. However, the present studies show that β-VLDL can be oxidized by Incubation with endothellal cells or with copper ions. Oxidized β-VLDL was degraded by macrophages at about twice the rate of unoxldized 0-VLDL, and It stimulated cholesterol estertflcatJon twice as much as unoxldized β-VLDL. The degradation of oxidized β-VLDL was Inhibited either by oxidized β-VLDL Itself or by oxidized LDL, but not by unoxldized β-VLDL 0-VLDL was chemotactic for human monocytes and contained significant amounts of lysophosphatldylcholine, previously shown to be a chemotactic agent In summary, oxidized LDL Is degraded by macrophages proportionately more than oxidized 0-VLDL as compared to the unmodified llpoprotelns. However, the twofold Increase may, nevertheless, be significant in the atherogenlclty of 0-VLDL.

A comparison of the antiatherogenic effects of probucol and of a structural analogue of probucol in low density lipoprotein receptor-deficient rabbits.

The efficacies of probucol and a close structural analogue as antioxidants in the prevention of atherogenesis in LDL receptor-deficient rabbits were compared. The antioxidant potency of the analogue in vitro was equal to that of probucol. Its biological availability was much greater: almost comparable concentrations in total plasma were achieved by feeding 1% probucol (wt/wt) and 0.05% analogue (wt/wt). Total plasma concentrations were comparable, but the concentration of probucol within the LDL fraction was about twice that of the analogue. Probucol slowed lesion progression by almost 50%, confirming earlier reports; the analogue, however, showed no detectable inhibitory effect on atherogenesis. Resistance of LDL to oxidation was measured at the end of the study by incubating it with Cu2+ and measuring the rate of diene conjugation. Probucol prolonged diene conjugation lag time from the control value of 130 min to values > 1,000 min. The analogue approximately tripled the lag time (mean, 410 min) and yet failed to slow the atherogenic process. The results suggest that LDL resistance to oxidation must reach some threshold level before there is significant protection against atherogenesis. However, probucol has additional biological effects, possibly not shared by the analogue, that could contribute to its antiatherogenic potential.

Interaction between High Density and Low Density Lipoproteins during Uptake and Degradation by Cultured Human Fibroblasts

High density lipoprotein (HDL) inhibited the binding (trypsin-releasable radioactivity), internalization (cell-associated radioactivity after trypsinization), and degradation (TCA-soluble non-iodide radioactivity) of (125)I-low density lipoprotein ((125)I-LDL) by cultured normal human fibroblasts. At HDL:LDL molar ratios of 25:1 (protein ratios about 5:1), these parameters were reduced by about 25%. Unlabeled LDL was about 25 times more effective in reducing (125)I-LDL binding, implying that if HDL and LDL bind at common sites the affinity of HDL for these sites is very low or that the interaction is on some other basis. The fractional reduction in (125)I-LDL binding at a given HDL: (125)I-LDL ratio was independent of (125)I-LDL concentration and occurred equally with fibroblasts from a subject with homozygous familial hypercholesterolemia. Reciprocally, the binding, internalization, and degradation of (125)I-HDL were reduced by LDL. Preincubation of fibroblasts with HDL (or LDL) reduced the subsequent binding of (125)I-LDL (or (125)I-HDL) during a second incubation. In other studies HDL reduced the net increase in cell cholesterol content induced by incubation with LDL. HDL alone had no net effect on cell cholesterol content. These findings suggest that HDL reduces both the high affinity and the low affinity binding of LDL to human fibroblasts and that this in turn reduces the internalization and degradation of LDL. The effect of HDL on the LDL-induced changes in cell cholesterol content could be in part on this basis and in part on the basis of an HDL-stimulated release of cholesterol from the cells. These effects of HDL in vitro may be relevant to the negative correlations reported from in vivo studies between plasma HDL concentration and both body cholesterol pool size and the prevalence of clinically manifest atherosclerosis but further studies will be needed to establish this.

Measurement in vivo of irreversible degradation of low density lipoprotein in the rabbit aorta. Predominance of intimal degradation.

The development of a highly sensitive method for assessing, tissue by tissue, the rates of irreversible protein degradation in vivo has allowed us to quantify low density lipoprotein degradation in the normal rabbit aorta and to localize it. The method depends upon the fact that tyramine-cellobiose, like sucrose used in previous studies, can be covalently attached to proteins, enter cells with them, and then remains trapped within the cell after the remainder of the protein molecule has been degraded. Rabbit LDL (d = 1.02 to d = 1.06 g/ml) was labeled with 125I-tyramine-cellobiose and injected into rabbits. Aortic 125I content 24 hours later served as a cumulative measure of degraded LDL (after appropriate corrections for any intact, nondegraded LDL present). Calculated aortic degradation of LDL averaged 9.4 X 10(-3) percent of the plasma pool per g aortic wet weight per day (n = 6). Intimal cells, obtained by gentle swabbing, accounted for fully 40% of total aortic degradation even though the intima represented less than 5% of the aortic mass. Autoradiography confirmed the high concentration of label in the intima. Degradation of unmodified and reductively methylated LDL were compared. The fractional rate of degradation of methylated LDL by the intima was 50% to 60% of that for native LDL, indicating that 40% to 50% of LDL degradation in the intima, predominantly endothelial cells, is mediated by LDL receptors.