Henry F. Hoff

Isolation, purification, and characterization of a lipoprotein containing Apo B from the human aorta.

Abstract LDL-like lipoproteins were extracted from a buffer-soluble fraction of homogenates of both grossly normal intima and fatty-fibrous plaques from the human aorta. A low-speed supernate of such homogenates was subjected to differential ultracentrifugation to isolate a d 1.006–1.063 density fraction which was further purified by gel filtration on agarose columns, or by affinity chromatography of low-speed supernatants of aortic homogenates on immunoabsorbents (anti-apo B). All recovered immunoreactivity for apo B as measured by electroimmunoassay was found in the retarded fraction following gel filtration or affinity chromatography. Characterization of the aorta-extracted lipoprotein containing apo B illustrated that the lipoprotein was quite similar to plasma LDL with respect to lipid composition, lipid/protein ratio, identification of apo B by polyacrylamide gel electrophoresis, and particle size by electron microscopy. However, aorta-derived lipoproteins differed from plasma LDL with respect to fatty acid composition and electrophoretic mobility. They showed an increase in stearate content which was most prominent in the cholesteryl ester and triglyceride fractions, as well as an increase in oleate and a reduction in linoleate content in both cholesteryl ester and phospholipid fractions. During purification aorta-derived lipoproteins maintained an increase in electrophoretic mobility compared to plasma LDL. This increase in surface charge might be a result of the demonstrated contamination with sulfated glycosaminoglycans (1.5% by weight). However, digestion with chondroitinase ABC had no effect on electrophoretic mobility. The lipoproteins extracted and purified from grossly normal intima and fatty-fibrous plaques possessed the same similarities as well as differences in characteristics relative to plasma LDL. The results from this study suggest that the lipoproteins extracted from the human aorta may represent either the preferential retention of a subclass of plasma LDL with slightly different characteristics from the average but with greater affinity for intimal material, or the products of modification of normal plasma LDL following retention by extracellular components of the aortic intima. This modification may be due to complex formation of LDL with such components.

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.

Localization of low-density lipoproteins in atherosclerotic lesions from human normolipemics employing a purified fluorescent-labeled antibody

Abstract The localization pattern of low-density lipoproteins in atherosclerotic arteries from normolipemic humans has been determined employing immunofluorescent techniques. Goat antiserum was prepared against delipidated human low-density lipoproteins (apo low-density lipoproteins) and conjugated with fluorescein isothiocyanate. A monospecific antibody was isolated by affinity chromatography using low-density lipoproteins attached to a column of Sepharose 4B. This antibody reacted with both low-density lipoproteins and apo low-density lipoproteins, and the fluorescence localization experiments did not distinguish between the lipoprotein and apoprotein. Sections of uninvolved areas, fatty streaks, and fibrous plaques from aorta, coronary, carotid and cerebral arteries from 20 normolipemics were studied following incubation with the purified antiserum. Uninvolved segments demonstrated no specific fluorescence. Fatty streaks and the fibromuscular caps of fibrous plaques demonstrated specific fluorescence to collagen and elastic fibers. Specific fluorescence was seen in fibrous plaques in large bands of collagen, in acellular areas as well as in the atheromatous core. Although this localization pattern was found consistently in arteries from normolipemics, not all atheromas or collagen and elastic fibers from these subjects were positive. By contrast, incubation of serial sections with fluorescein isothiocyanate-conjugated non-immune globulins resulted in non-specific fluorescence to only the cytoplasm of all intimal and medial cells. This non-specific pattern was present in both non-involved and involved segments of arteries.

Interaction of phosphatidylcholine and apolipoprotein-alanine: electron microscopic studies☆

Abstract Protein-lipid complexes of apolipoprotein-alanine and phosphatidylcholine were studied by electron microscopy using the technique of negative staining. An unfractionated sonicated dispersion of phosphatidylcholine vesicles of varying size was combined with apolipoprotein-Ala without exposure to further sonication. The resulting complexes appeared as linear arrays of stacked discs varying in length from 500 to 5000 A. These stacks appeared to form by two processes: (1) by the transverse cleavage of segments of discs from multilamellar vesicles; and (2) by alignment of bilamellar vesicles into linear arrays. When fractionated bilamellar vesicles of uniform diameter (250 A) were titrated with increasing amounts of apolipoprotein-Ala, the vesicles underwent a series of morphological changes from free vesicles to stacked discs to parallel groups of stacked discs to myelin figures.

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.

Apo B concentration in the normal human aorta

Abstract The concentration of LDL-protein (apo B) was determined by electroimmunoassay in the grossly normal intima of 15 human aortas obtained at autopsy. The mean apo B concentration was: 1.02 ± 0.05 SEM (range 0.16 – 2.51) mg per cm 3 tissue, or higher than literature values of plasma apo B. No detectable amounts of apo B were found in the neighboring tunica media in any of the cases. In one case apo B concentrations were also measured in tissues from liver, lung, spleen, kidney, brain, myocardium and skeletal muscle, but all had values at least ten-fold lower than aortic intima, except liver (one-fourth intima value). The mean concentrations of human serum albumin (HSA) in the intima was 13.52 mg/cm 3 , or only one-fourth plasma concentration. Thus the aortic intima not only has the highest apo B values of the tissues tested, but in the intima apo B is retained to a greater degree than another plasma macromolecule. These results may be relevant to the fact that arterial intima is the primary site of atherogenesis.

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.

Ultrastructural Localization of Plasma Lipoproteins in Human Intracranial Arteries

The fine-structural localization of apoB, the major protein constituent of both the low and very low density plasma lipoprotein fractions, was described in human middle cerebral and basilar arteries. Using an immunoperoxidase technique together with electron microscopy, apoB was localized only in arteries with atherosclerotic involvement and to the following regions in these arteries: 1. on the outer aspects of extracellular spherical structures with diameters of 250 to 700 Å found predominantly in lipid cores and between bands of collagen fibers of advanced atherosclerotic lesions; 2. on the surface of reduplicated elastica; 3. along collagen fibers and; 4. on aggregates of extracellular spherical lipid globules. These results suggest that the extracellular spheres may represent the fine-structural morphology of deposited low and very low density lipoproteius and that free apoB may be bound to lipid globules, elastica, and collagen fibers.