Carol L. Heideman

Localization patterns of plasma apolipoproteins in human atherosclerotic lesions.

The localization patterns of human plasma lipoproteins and their respective apoproteins and of neutral lipid were determined in normal and atherosclerotic arteries. Specific antisera were prepared against plasma low-density lipoproteins (LDL) and their apoproteins (apoB), high-density lipoproteins (HDL) and one of their major apoproteins (apoA-I), and apoC-III, which is a major apoprotein of very low-density lipoproteins (VLDL). Using immunofluorescence techniques, the various antigens were localized in arterial samples obtained at surgery or autopsy. The three apoproteins and neutral lipid were localized to the same tissue areas, namely, lipid core regions and certain connective tissue of atherosclerotic lesions, in 61% of the fibrous plaques and 48% of the fatty streaks examined. In marked contrast, none of the uninvolved arterial regions showed the presence of all four factors together. As controls, the localization of other serum proteins was also determined in these arteries using immunofluorescence techniques. Fibrinogen was associated with regions of maximum complementary localization of factors in 37% of the fibrous plaques and 64% of the fatty streaks. However, albumin was found in only 4−5% of these same regions. The present results suggest that not only LDL but also HDL and VLDL or their respective apoproteins as well as fibrinogen are specifically retained by certain tissue components of the atherosclerotic lesion.

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.

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

SUMMARY Apolipoprotein B (apoB) was measured in buffer-extracted homogenates of grossly normal and atherosclerotic human aortic intima by means of an electroimmunoassay procedure. The apoB values which were expressed as μg 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. Bufferextractable 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.

Quantification of apolipoprotein B in grossly normal human aorta.

SUMMARY A quantitative electroimmunodiffusion (EID) assay was developed for apolipoprotein B (apoB), the major apoprotein of human plasma low density lipoproteins (LDL) and very low density lipoproteins (VLDL). Specificity, sensitivity (30–200 ng) and reproducibility (11%) were established. We used this system to determine the amount of buffer-soluble apoB in supernatant fractions from homogenates of the intima from grossly normal human aortas. Assays of whole tissue minces yielded only one-third of the apoB in supernatant fractions. Intimal homogenates contained 0.34–18.45 &mgr;g of apoB/mg of tissue, dry weight (mean 5.42 ± 3.95 SD). We also found that no apoB was detected in the homogenates of adjacent tunica media. Furthermore, most of the intimal apoB was found in the LDL density (d) fraction (d 1.006–1.063) after differential ultracentrifugation, while the VLDL (d < 1.006) fraction contained only negligible amounts of apoB. By electron microscopy, it was determined that the LDL density fraction contained particles 200–250 A in diameter which were similar in size to plasma LDL. These results suggest that grossly normal aortas contain significant quantities of intact LDL.

Correlation in the human aorta of APO B fractions with tissue cholesterol and collagen content.

The amounts of buffer- and Triton-extracted apo B (LDL-protein), as well as the sum of these two fractions, were correlated with the total tissue cholesterol and hydroxyproline content (as a measure of collagen) in grossly normal intima, fatty streaks, and fibrous plaques of human aortas obtained at autopsy. Quantitative values of buffer- and Triton-extracted apo B were obtained by sequentially extracting homogenates of aortic intima with an aqueous buffer and one containing Triton X-100, and measuring the apo B content in each extract by an electroimmunoassay relative to plasma LDL or Triton-treated LDL. Significant positive correlations were obtained between the following: tissue cholesterol and both buffer-extracted and total-extracted apo B in grossly normal intima; tissue cholesterol and Triton-extracted apo B in microdissected fibrotic caps and cores of fibrous plaques, as well as in whole plaques. A positive correlation was also obtained between tissue cholesterol and total-extracted apo B in the necrotic core. A significant negative correlation was found between Triton-extracted apo B and collagen in whole plaques. The calculated mean percent of total tissue cholesterol in the different aortic regions that could be present as part of an intact LDL particle were: 100% in grossly normal intima, 16% in fatty streaks, and 11% in fibrous plaques. The positive correlation between Triton-extracted apo B and cholesterol in plaques suggests one or both of the following: the extracellular pool of cholesterol or some material increasing concurrently with cholesterol interacts with apo B or another part of the LDL particle; or the apo B containing lipoprotein is trapped in the hydrophobic environment of extracellular lipid. Both possibilities would render the particle less soluble in aqueous buffers. The negative correlation between Triton-extracted apo B and tissue collagen and the lack of a significant correlation between buffer-extracted apo B and collagen content suggests that collagen is probably not responsible for apo B retention in the aortic intima.

Detergent extraction of tightly-bound apoB from extracts of normal aortic intima and plaques

Abstract Previous studies have suggested that aortic plaques contain a substantial quantity of LDL-protein or apoB not removed by conventional buffer extraction. We have examined the extraction of this remaining apoB from pellets of aortic intimal homogenates under a variety of conditions. Triton X-100 was the most effective of five detergents listed for their ability to extract residual apoB. This fraction was quantified by an electroimmunoassay (EIA) relative to an LDL standard treated with Triton. Standard slopes of peak height versus concentration were flatter for Triton-treated LDL than native LDL. On Ouchterlony plates lines of complete identity were obtained between native LDL, Triton-treated LDL, and buffer-extracted apoB, but only partial identity between Triton-extracted residual apoB and the above. However, when whole minces of aortic plaques were homogenized and extracted with Triton, the amount of apoB obtained was comparable to the sum of buffer-extractable and detergent-extractable apoB. When exogenous Triton-treated LDL was added to aortic pellets or to supernatant fractions of Triton-extracted pellets, recovery appeared to be complete within the error of the system. Extraction of residual apoB from plaque pellets with Triton appeared complete since no apoB could be detected by immunofluorescence techniques in the pellets remaining after three extractions with Triton or by EIA in the third extract. In twelve cases studied normal intima contained predominantly buffer-extracted apoB, while plaques contained nearly equal amounts of buffer- and Triton-extracted apoB. These results suggest that the plaques contain a tightly bound fraction of apoB in contrast to the normal intima.

Lipoproteins containing apolipoprotein A-I extracted from human aortas☆

Apolipoprotein A-I was quantitated by electroimmunoassay in buffer-soluble fractions of both grossly normal intima and raised atherosclerosis lesions of the human aorta. The mean value for apolipoprotein A-I content in microgram/mg tissue dry weight of normal intima (12 cases) was 0.71 +/- 0.10 S.E. and of aortic plaques (19 cases) was 0.64 +/- 0.40 S.E. When compared to the buffer-extractable apolipoprotein B content measured in these same cases from both regions, the ratio of apolipoprotein B to apolipoprotein A-I was approximately 6. No apolipoprotein A-I was measurable in tunica media. Following differential ultracentrifugation into d less than 1.063, d 1.063-1.21 and d greater than 1.21 fractions, the distributions of recovered apolipoprotein A-I were, respectively: 1, 94 and 5% for normal intima, 19, 31 and 50% for plaques and 1, 89 and 10% for plasma. Characterization of a chromatographically purified d 1.063-1.21 or HDL density fraction from fatty-fibrous plaques demonstrated particles of between 60 and 120 A diameter, a characteristic apolipoprotein A-I band by SDS-polyacrylamide gel electrophoresis, and a precipitin peak closely migrating with that for plasma HDL by two-dimensional immunoelectrophoresis. The d greater than 1.21 density fraction from plaques isolated by affinity chromatography on a Sepharose-anti-apolipoprotein A-I column contained small amounts of phospholipid but no measurable cholesterol. The d 1.063-1.21 density fraction from plaques showed a significant increase in percent free cholesterol and phospholipid contents and decrease in cholesteryl ester content relative to plasma HDL. This increase in free cholesterol could represent evidence for an anti-atherogenic mechanism wherein infiltrated HDL removes cholesterol together with phospholipid from the arterial wall.

Quantitation of apo b in human aortic fatty streaks A comparison with grossly normal intima and fibrous plaques

Abstract The content of apolipoprotein B (apo B)-containing lipoproteins was measured in aortic fatty streak lesions of 18 male individuals between the ages of 21 and 67 years, and compared to the values found in adjacent grossly normal intima. Extraction of apo B was accomplished by sequential treatment of aortic tissue homogenates with a standard buffer and one containing the detergent Triton X-100. Mean apo B values (μg per mg tissue dry weight) in fatty streaks were: buffer-extracted = 4.67 ± 0.51, Triton-extracted = 1.88 ± 0.39; while in adjacent grossly normal intima: buffer-extracted = 6.51 ± 0.92, Tritonextracted=0.37 ± 0.15. Using a paired t -test, buffer-extracted apo B was marginally significantly greater in the adjacent normal intima than fatty streaks ( P P t -test, buffer-extracted apo B was slightly higher in fatty streaks than fibrous plaques ( P P

Localization of Apo-Lipoproteins in Human Carotid Artery Plaques

Apoproteins from plasma lipoproteins were localized by immunofluorescence techniques in human carotid artery atherosclerotic lesions. These studies were performed in light of the possible importance of these apoproteins in both lipid metabolism and the pathogenesis of atherosclerosis. ApoA-I from high density lipoproteins, apoB from low density lipoproteins, and apoC-III from very low density lipoproteins were localized also as markers for their respective lipoproteins, since the latter cross-react immunologically. The three apoproteins were localized to the same regions of lesions as neutral lipids and, to some extent, fibrinogen. These regions consisted of bands of collagen fibers, usually deeper within the lesion, and the lipid core or atheroma of such advanced lesions. Although the superposition of localization for the three apoproteins and lipid was only 53%, it was suggested that deviation from complete superposition was due to the abrupt changes in lesion structure resulting from the focal nature of the atherosclerotic process. These results suggest that there is a broader specificity than previously implied of the interaction between such lesion components as connective tissue and extracellular lipid accumulations, and apoproteins from plasma lipoproteins. This interaction is believed to result in a net retention within atherosclerotic lesions of human extracranial arteries of these plasma-derived factors, either as free apoproteins or as native lipoproteins.

Low Density Lipoproteins in the Aorta: Relation to Atherosclerosis

A link between plasma low density lipoproteins (LDL) and the atherosclerotic process has been suggested from both clinical-correlative studies in man (Kannel et al. 1964) and diet-fed experimental animal models (St. Clair 1976). LDL is the major carrier of cholesterol into the arterial wall and the deposition of cholesterol is the hallmark of the atherosclerotic process (St. Clair 1976). However the details surrounding the transport and deposition of cholesterol are not completely understood. LDL could transit the entire vessel wall and enter the lymphatics. It could be metabolized following uptake by arterial smooth muscle cells as was shown in cultured fibroblasts (Goldstein and Brown 1977). In a non-human primate model as much as 25% of total LDL entering the aorta became TCA-soluble, suggesting breakdown of the protein moiety (Hollander et al. 1977). Finally some LDL could be selectively retained in the arterial intima. A retention of this lipoprotein in the intimai lining of major arteries has been demonstrated by numerous immuno- fluorescence studies from our laboratory (Hoff et al. 1974, 1975a,b).