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Quantitation of apo b in human aortic fatty streaks A comparison with grossly normal intima and fibrous plaques
263
Atherosclerosis, 30 (1978) 263-272 Scientific @ Elsevier/North-Holland
QUANTITATION
Publishers,
Ltd.
OF APO B IN HUMAN AORTIC FATTY STREAKS
A Comparison with Grossly Normal Intima and Fibrous Plaques HENRY F. HOFF, CAROL L. HEIDEMAN, ANTONIO M. GOTTO, JR. Departments (U.S.A.)
JOHN W. GAUBATZ,
JACK L. TITUS and
of Medicine and Pathology, Baylor College of Medicine, Houston, TX 77030
(Received 12 December, 1977) (Revised, received 20 March, 1978) (Accepted 19 April, 1978)
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 (E.cgper 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 f 0.15. Using a paired t-test, buffer-extracted apo B was marginally significantly greater in the adjacent normal intima than fatty streaks (P < 0.05), whereas Triton-extracted apo B was highly significantly greater in fatty streaks than adjacent normal intima (P < 0.0005). When the mean apo B values of these 18 fatty streaks and 23 fibrous plaques from separate cases were compared in a non-paired t-test, buffer-extracted apo B was slightly higher in fatty streaks than fibrous plaques (P < 0.025), whereas Triton-extracted apo B was much higher in fibrous plaques than in fatty streaks (P < 0.0005). The intermediate position of fatty streaks between grossly normal and fibrous plaques with respect to buffer- and Triton-extracted apo B content, gives additional support to the contention that this lesion is an intermediate step in the progression of the grossly normal intima to a fibrous plaque. Assuming this sequence of progression to occur, our results demonstrate a marginally signifiSupported by the National Heart and Blood Vessel Research and Demonstration Center, Baylor College of Medicine (NIH Grant HL-17269). NIH Grant NS-09287 and a Grant in Aid from the American Heart Association. Dr. Hoff is an Established Investigator of the American Heart Association. Send reprint requests to: Dr. Henry F. Hoff. Department of Medicine, Baylor College of Medicine, Houston, TX 77030. U.S.A.
264
cant decrease in buffer-extracted apo B but a highly statistically increase in Triton-extracted apo B with lesion development. Key words:
Aortas - Apolipoprotein B (APO B) - Atherosclerosis -Low density lipoproteins -Man
significant
- Electroimmunoassay
Introduction In the progression of atherosclerosis in man the fatty streak lesion has long been considered to be the precursor of the fibrous plaque [ 11 as defined by WHO criteria [ 21. However, in recent years this point has become increasingly controversial [ 1,3]. Supportive of this contention are data from experimental animal models in which atherosclerosis is induced by feeding diets high in cholesterol and saturated fats [4,5]. Early lesions in animals resemble the human fatty streaks and later appear as a more advanced lesion usually resembling human fibrous plaques [ 1,4,5]. Changes intermediate between the two lesions have been documented in human arteries by conventional morphology [6], enzyme histochemistry [ 71, and physical chemistry of the lipids in the lesions [8]. On the other hand, while both lesions are located at the same anatomic sites of coronary arteries in man [ 1,3,9], fibrous plaques of the aorta in a later decade of life usually do not develop at the same geographic locations where fatty streaks are found during the second and third decades of life [lo]. Moreover, the major fatty acid in the cholesterol ester fraction of fatty streaks is oleic acid whereas in fibrous plaques it is linoleic acid [ 11,121, as it is in plasma low density lipoproteins (LDL) [ 131. In a recent study, we quantitatively measured apolipoprotein B (apo B) the major protein of LDL, in both the grossly normal aortic intima [14] and in aortic fibrous plaques [ 151. Most of the accumulated apo B in the normal intima is extractable from a tissue homogenate with a standard buffer or saline [ 141. However, in aortic plaques only half of the apo B is extractable with a conventional buffer, the remainder extractable with the detergent Triton X-100 [16]. Conceivably, if fatty streaks are precursors of the fibrous plaque, then the amount of apo B present in buffer- or Triton-extractable forms would be intermediate between grossly normal intima and aortic plaques. In this report we have documented the amounts of buffer- and Triton-extracted apo B in 18 human aortic fatty streaks and demonstrated that these values lie intermediate between those found in adjacent normal intima and those found in 23 fibrous plaques from separate cases. Material and methods Each aortic sample was obtained at autopsy from the County Medical Examiner within 18 h after death. Most cases were trauma victims. Fatty streak lesions and adjacent grossly normal regions were excised from the descending thoracic aorta from 18 males between the ages of 21 and 67 years. Fibrous plaques [2] were also removed from the aorta of 23 males between the ages of 39 and 72 years. Grossly normal regions located between the plaques were
265
present in 10 of the 23 cases and were also removed. The size of the intimal surface of grossly normal intima removed ranged from 10 to 30 cm2 depending on the thickness of the intima. The intimal lining from each tissue specimen was stripped from the underlying tunica media, finely minced, and homogenized in a standard Tris-EDTA buffer pH 7.4 using a Polytron homogenizer (Brinkmann, New York) as described in detail earlier [ 14,151. Aliquots of the supernatant fraction of this homogenate were measured for buffer-extracted apo B content using an electroimmunoassay (EIA) [ 14,171. The pellets of such homogenates extracted twice with buffer were re-extracted with 3% Triton X-100 in the standard Tris buffer to remove any residual apo B which could not be extracted with a standard buffer. Aliquots of this supernatant fraction were applied to an EIA using LDL treated with Triton as a standard. The details of the procedure to quantitate the Triton-extracted apo B fraction have been reported elsewhere [ 161. Representative regions of each sample of grossly normal intima, fatty streak, or fibrous plaque were taken both for conventional microscopy, i.e. Movatstained [18] sections of paraffin-embedded specimens, and for cryostat sections which were stained for lipid with oil red 0 and apo B with an immunofluorescence procedure [ 19,201. A Leitz Orthoplan photomicroscope was employed in conjunction with a Leitz Ploem illumination system for fluorescence microscopy and interference contrast system for bright field microscopy. Results
General morphologic description The smooth-surfaced intimal lining of the grossly normal human aorta from adults demonstrated thickness varying from approximately 50-150 I.tm. As shown previously the intima was characterized by the presence of smooth muscle cells in a matrix of collagen fibers and proteoglycans with reduplicated or fragmented elastica present at the intima-media border [ 141. Lipid was also localized in this area usually associated with connective tissue fibers. Apo B was often present diffusely localized in the thickened intima of adults, but not in the tunica media [14]. Fatty streak lesions from adults were characterized grossly as small, slightly raised yellow regions, surrounded by the smoother and whiter grossly normal intima (Fig. la). Microscopically this lesion demonstrated intimal thickening, slightly greater than corresponding grossly normal areas (90-200 pm). Numerous foam cells with both round and spindle shapes were present in the intima, as well as monocytes (Fig. lb, c), collagen fibers, proteoglycans and some reduplicated or fragmented elastic fibers (Fig. Id). Oil red O-stainable lipid was present mainly within foam cells, but also extracellularly (Fig. le), whereas apo B was present diffusely in the extracellular space (Fig. If). Pellets of homogenates of either fatty streaks or normal intima were negative for apo B by immunofluorescence following two extractions with Triton. As demonstrated in earlier studies the grossly raised fibrous plaque usually had a necrotic core containing lipid gruel demonstrable by oil red 0 stain at its base and a fibrotic or fibromuscular cap on the lumen surface [2,15]. The topographical distribution of apo B, as seen in earlier studies [19,20] was
266
Fig. 1. (I: gross morphology of fatty streaks in human thoracic aorta from a 23-year-old individual. Note the rows of small raised areas. X1.2. b: micrograph of a Movat-stained section of a fatty streak lesion using interference contrast optics. Note the presence in the intima of numerous foam cells and blood monocytes. L = lumen. X120. c: higher magnification of fatty streak depicting the vacuolated appearance of the foam cells close to the lumen border. Interference contrast optics. L = lumen. X 300. d: micrograph of the intima-media border of a fatty streak lesion depicting the reduplicated elastica (black lines) on the intimal side together with numerous collagen fibers seen as bundles of wavy material. Some of the bundles stained positively with alcian blue suggesting the association of glycossminoglycans with some of the collagen. X300. e: oil red O-stained cryostat section of a fatty streak. The dark droplets depicting lipid can be seen as large spheres close to nuclei. presumably representing intracellular lipid, as well as extracellularly localized smaller spheres (arrow). X180. f: localization of apo B (white) only in the intima of this fatty streak. Elastic fibers in media autofluorescent. Immunofluorescence stain. L = lumen. X 100.
found often together with oil red O-stainable lipids as a diffuse array in the necrotic core and along connective tissue fibers in the fibromuscular cap. For quantitative studies we utilized only one plaque from each case, and only lesions which by microscopy demonstrated lipid-filled necrotic cores which encompassed over 50% of the total cross-sectional area [ 211.
267
Quan titation of apo B The buffer- and Triton-extracted apo B content in c(g per mg tissue dry weight of fatty streaks from the thoracic aortas of 18 individuals was compared to values obtained from the adjacent grossly normal intima (Table 1). By statistical analysis (paired t-test) the buffer-extracted apo B content in the grossly normal intima (mean value: 6.51 f 0.92) was only marginally significantly greater (P < 0.05) than the corresponding value in fatty streaks (mean value: 4.67 + 0.51). In cast 5 cases (2, 3, 4, 8 and 15) showed the opposite trend. TABLE 1 BUFFERGROSSLY Case NO.
AND TRITON-EXTRACTED NORMAL INTIMA Age/ Race
Sample a
2
21w 21w
3
21b
4
22w
5
23b
6
26~
7
27~
8
29w
9
29w
10
33w
11
33b
12
37b
13
39w
14
41w
15
44b
16
52~
17
57w
18
67~
Mean f SEM
34 + 3
--
N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS
FATTY
STREAKS
AND
ADJACENT -
Triton-extracted apo B
Buffer-extracted ape B Wmg
1
APO B IN AORTIC
D.W.
5.65 2.01 0.92 4.07 0.55 2.50 0.75 2.81 7.72 7.05 9.23 7.47 6.56 4.73 2.78 9.36 7.00 3.97 11.98 7.41 5.90 4.66 3.63 1.40 9.92 2.82 11.38 5.19 2.48 3.87 10.83 3.37 10.82 6.26 9.26 5.19 6.51 + 0.92 4.67 f 0.51 e P < 0.05
9%of total
Wmg
100 100 100 100 57 100 45 90 74 83 78 84 61 70 65 100 a4 100 96 100 88 100 100 100 54 90 50 100 61 100 81 100 95 100 62 94.6 75.1
0 0 0 0 0 1.91 0 3.42 0.90 2.41 1.94 2.01 1.20 3.00 1.16 5.03 0 0.76 0 0.21 0 0.65 0 0 0 2.40 1.41 5.20 0 2.43 0 0.81 0 0.30 0 3.14 0.37 f 0.15 1.88 f 0.39 o P < 0.0005
.
D.W.
% of total 0 0 0
0 0 43 0 55 10 26 17 22 16 39 30 35 0 16 0 4 0 12 0 0 0 46 10 50 0 39 0 19 0 5 0 38 5.4 24.9
a N = grossly normal intima; FS = fatty streak. b In a paired f-test differences between normal and fatty streak group were statistically significant at the levels indicated.
268
However, in a paired t-test the Triton-extracted apo B content of fatty streaks (mean value: 1.88 f 0.39) was highly significantly greater (P < 0.0005) than that of adjacent normal areas (mean value: 0.37 + 0.15). The grossly normal intima of 13 of the 18 cases did not contain measurable amounts of Tritonextracted apo B. Numerous fatty streaks contained Triton-extracted apo B values over 3 (ug per mg tissue dry weight (cases 4, 7, 8, 14 and 18). In some of these lesions patchy areas of necrosis were seen by microscopy. Extracellular lipid and apo B distribution was also more widespread in fatty streaks from older individuals than younger ones. Although the width of each thickened intima increased with age in the grossly normal intima, no positive correlation could be found between either buffer- or Triton-extracted apo B per unit dry weight and intimal thickening in either normal intima or fatty streaks. We also compared the buffer- and Triton-extracted apo B content of the fatty streaks with that of fibrous plaques from a separate group of cases (Table 2). The mean buffer-extracted apo B content of the 18 fatty streaks (4.67 + 0.51) was higher than that of 23 fibrous plaques (3.18 + 0.41), but using a nonpaired t-test the level of statistical significance was marginal (P < 0.025). By contrast the mean Triton-extracted apo B content of the fibrous plaques (3.78 + 0.31) was highly significantly greater than that of fatty streaks (1.88 + 0.39). The level of significance was P < 0.0005. Finally in a series of grossly normal intimas comprising both the 18 normal regions adjacent to the fatty streaks plus 10 additional cases, we compared the buffer- and Triton-extracted apo B content from cases 39 years of age or above (>39) with those below 39 years of age (<39). This age was chosen because cases 39 years or over demonstrated the presence of raised aortic fibrous plaques, whereas the cases below 39 years of age did not demonstrate the presence of such lesions. As seen in Table 3, the normal intima of the group >39 years had mean buffer-extracted apo B values (7.66 f 0.84) marginally siginificantly greater (P < 0.05) than that of normal intima of the group <39 years (5.12 f 0.96). No significant differences were detected for Triton-extracted apo B values between the two groups. Comparing normal and atherosclerotic intima for the amount of apo B in loosely- and tightly-bound fractions (Tables 1 and 2) permitted the following statistically significant observations: for buffer-extracted apo B fractions, grossly normal intima (239 years) > grossly normal intima (<39 years) > fatty
TABLE 2 COMPARISON OF THE MEAN BUFFER- AND MEAN FATTY STREAKS AND FIBROUS PLAQUES Lesion
Fatty streak Fibrous plaque
Mean age f SEM
34 + 3 56 f 4
Number of cases
18 23
TRITON-EXTRACTED
APO B VALUES
Mean apo B value (pg per mg tissue dry weight) f SEM Buffer-extracted
T&on-extracted
4.67 f 0.51 a 3.18 + 0.41 a
1.88 * 0.39 b 3.78 + 0.31 b
a Statistically significant difference using non-paired t-test: P < 0.025. b Statistically significant difference using non-paired t-test: P < 0.0005.
IN
269 TABLE 3 COMPARISON OF THE MEAN BUFFER- AND MEAN TRITON-EXTRACTED APO B VALUES IN THE GROSSLY NORMAL AORTIC INTIMA FROM CASES BELOW AND ABOVE 39 YEARS OF AGE Mean age 2 SEM
Number of cases
Mean ape B value (pg per mg tissue dry weight) f SEM
Normal (>39 Normal (<39
yrs) yrs)
52 + 3 27 + 2
15 13
Buffer-extracted
Triton-extracted
7.66 i 0.84 a 5.12 i- 0.96 a
0.16 i- 0.10 b 0.43 f 0.20 b
a Differences statistically significant: P < 0.05. b Differences not statistically significant.
streaks > fibrous plaques; for Triton-extracted apo B fractions, fibrous plaques > fatty streaks > normal intima. It should be noted that only for the Tritonextracted apo B groups were the differences highly statistically significant (P < 0.0005). Discussion We have compared the apo B content of human fatty streaks from the thoracic aorta of 18 males with that of adjacent grossly normal intima as well as with that of fibrous plaques from 23 separate cases. Smith et al. [22,23] had previously compared values of apo B (expressed as LDL cholesterol) in human aortic fatty streaks with those in adjacent grossly normal intima. They also found lower values for apo B in fatty streaks than in adjacent normal intima, as we have shown, but the differences were much greater than in our study. The discrepancy in results could be due to the differences in techniques employed i.e. Smith et al. [22,23] inserted minces of aortic intima directly into wells of EIA, whereas we pipeted ahquots of supernatant fractions of tissue homogenates, a procedure we have shown to measure more apo B than when applying minces directly to the assay. The discrepancy could also be due to the preferential dissection by Smith et al. of focal regions of fatty streaks containing large accumulations of foam cells essentially free of extracellular material. By contrast the tissue used by us in this study contained both regions filled with foam cells as well as large regions of extracellular space. Recently Smith et al. [24] demonstrated that a substantial amount of residual apo B could be measured from minces of raised atherosclerotic lesions which had previously been applied to an EIA. This was accomplished by treating aortic minces with such hydrolytic enzymes such as plasmin or collagenase and then re-assaying. In some plaques the residual apo B was about the same amount as the buffer-extracted fraction of apo B [ 241. It is quite possible that residual apo B was also present in the minces of fatty streaks, which would correspond to the Triton-extracted apo B fraction in our study. In a previous report we have presented evidence demonstrating that the combined treatment of arterial homogenates with a conventional buffer, followed by one containing Triton, quantitatively removed all apo B from the arterial tissue [ 161. In those studies we had shown that grossly raised fibrous
270
plaques contain roughly equal amounts of buffer- and Triton-extracted apo B, whereas adjacent grossly normal intima contained primarily buffe;-extracted apo B. In this present study, fatty streak lesions contained significantly higher values of residual apo B, extractable with Triton X-100, than adjacent normal areas. Fatty streaks were intermediate between grossly normal intima and fibrous plaques with respect to both buffer- and Triton-extracted apo B. These findings suggest that the fatty streak may be an intermediate step in the progression from a grossly normal intima to a fibrous plaque [ 1,3]. Differences in topographical distribution of fatty streaks earlier and fibrous plaques later in life on the aortic lumen surface [ 1,3,10] do not necessarily preclude a precursor-product relationship between the two. They merely suggest that some fatty streaks may develop into fibrous plaques while others regress. Significant correlations, however, were found between the appearance of aortic fatty streaks in the second decade of life containing small amounts of necrosis or monocytes and the appearance of fibrous plaques in the same location after the fourth decade [ 251. Differences in the fatty acid pattern of cholesterol esters in the fatty streaks and fibrous plaques [11,12] has been used as evidence refuting a precursorproduct relationship between these two lesions [ 1,3]. However, greater retention of the linoleic acid-rich plasma LDL by the fatty streak as it progresses might have diluted out the oleic acid-rich content of the original fatty streak which contains predominantly intracellular lipid. Katz et al. [S] have demonstrated the occurrence of aortic lesions in man in which the physical-chemical state of the lipid was intermediate between that found in fatty streaks and fibrous plaques. Such lesions contained both foam cells and extra-cellular pools of lipid. The quantitative results of apo B retention in this study suggest that, as an atherosclerotic lesion develops, so does the percent of total apo B which must be extracted with Triton. This is consistent with the results of an earlier study from this laboratory showing that the lipid core at the base of advanced lesions was the major region of Triton-extracted apo B in such plaques [ 221. When we compare the values in this present study with those obtained in the earlier study on plaque lipid cores and fibrotic capsules separated by microdissection, we find that lipid cores have the highest content of Triton-extracted apo B per unit dry weight. Also, even though most apo B in the fibrotic capsule was loosely-bound, the amount of Triton-extracted apo B in these capsules was greater than that in fatty streaks even though the difference was only significant at the P < 0.05 level. The finding that the Triton-extracted fraction of apo B in fatty streaks was larger than that of grossly normal intima from cases over 39 years of age suggests that the retention of this fraction of apo B is diseaserather than age-related. Little is yet known about the characteristics of the Triton-extracted fraction of apo B. It seems unlikely that the material is insoluble in aqueous buffer due to delipidation of apo B, since extracellular lipid is plentiful in both fatty streaks and grossly normal intima. Moreover, we have been able to liberate aqueous soluble apo B from pellets of plaque homogenates by digesting with elastase (unpublished observations) instead of treatment with Triton. Similar approaches were taken by Smith et al. [24] and Hollander [25]. These results
271
suggest that a portion of the Triton-extracted apo B was at least partially lipidated. Further preliminary data [27] has demonstrated significant positive correlations between this fraction of apo B and tissue cholesterol. Similar results were obtained by Smith et al. [24]. These data suggest the possibility that cholesterol is involved in the tight-binding of certain of the apo B-containing lipoproteins which insudate into the arterial wall. Further studies are required before the mode of apo B retention in the arterial wall is completely elucidated. Acknowledgments The authors wish to acknowledge the help of members of the Harris County Medical Examiner’s Office in aiding in the procurement of the aortic samples, and Dr. Henry C. McGill, Professor of Pathology, University of Texas Health Science Center, San Antonio, for constructive criticism in the preparation of the final manuscript. References 1 McGill, Jr., H.C., The lesion in atherosclerosis. In: G. Schettler and A. Weizel (Eds.), II Proc. of the Third International Symposium, Springer-Verlag, New York, 1974, pp. 27-38. 2 Classification of Atherosclerotic Lesions. Report of a study group: WHO Tech. Rep. Ser., 143 (1958) 3. 3 McGill, Jr., H.C., Atherosclerosis -Problems in pathogenesis, Atheroscler. Rev., 2 (1977) 27. 4 Clarkson, T.B. and Lofland, H.B., Effects of cholesterol-fed diets on pigeons susceptible and resistant to atherosclerosis, Circ. Res., 9 (1961) 106. 5 Scott, R.F., Daoud. AS. and Florentin. R.A.. Animal models in atherosclerosis. In: R.W. Wissler and J.C.Geer (Eds.), The Pathogenesis of Atherosclerosis. Williams and Wilkins, Baltimore, MD, 1972. pp. 120-146. 6 Geer, J.C.. McGill, Jr., H.C., Robertson. W.B. and Strong, J.P., Histologic characterization of coronary artery fatty streaks, Lab. Invest.. 18 (1968) 565. 7 Hoff, H.F., A histoenzymatic study of human intracranial atherosclerosis, Amer. J. Path., 67 (1972) 583. 8 Katz. S.S., Shipley, G.G. and lesions - Demonstration of a Invest., 58 (1976) 200. 9 Montinegro, M.R. and Eggan, 18 (1968) 586. 10 Mitchell, J.R.A. and Schwartz, 11 12 13
14 15 16
17 18 19
Small, D.M., Physical chemistry of the lipids of human atherosclerotic lesion intermediate between fatty streaks and advanced plaques, J. Clin. D.A.,
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Smith, E.B.. The influence of age and atherosclerosis on the chemistry of aortic intima. Part 1 (The lipids), J. Atheroscler. Res., 5 (1965) 224. Insull, Jr., W. and Bartsch. G.E., Cholesterol, triglyceride and phospholipid content of intima, media, and atherosclerotic fatty streak in human thoracic aorta, J. Clin. Invest., 43 (1966) 513. Skipski. V.P.. Lipid composition of lipoproteins in normal and diseased states in blood lipids and lipoproteins. In: G. Nelson (Ed.), Quantitation, Composition and Metabolism, Wiley-Interscience, New York, NY, 1972, p. 471. Hoff, H.F., Heideman, C.L.. Gaubatz, J.W., Gotto, A.M., Erickson, E.E. and Jackson, R.L., Quantitation of apolipoprotein B (apo B) in grossly normal human aorta, Circ. Res., 40, (1977) 56. Hoff, H.F., Heideman, C.L., Gaubatz, J.W. and Gotto, A.M., Apolipoprotein B (ape B) retention in the grossly normal and atherosclerotic aorta. Circ. Res., 41 (1977) 684. Hoff, H.F., Heideman, C.L., Gaubatz, J.W., Scott, D.W. and Gotto. A:M., Detergent extraction of tightly-bound ape B from extracts of normal aortic intima and plaques, Exp. Mol. Path., (1978) In press. Laurell, C.B.. Electrohnmunoassay, Stand. J. Clin. Lab. Invest.. 29 (Suppl. 124) (1972) 21. Movat, H.Z.. Demonstration of all connective tissue elements in a single section - Pentachrome stains. Arch. Path., 60 (1955) 289. Hoff. H.F., Jackson, R.L., Mao. J.T. and Gotto. A.M.. Localization of low density lipoproteins in
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phys. Acta. 351 (1974) 407. Hoff, H.F., Lie., J.T., Titus, J.L.. Bayardo, R.J., Jackson. R.L.. DeBakey, M.E. and Gotto, A.M., Lipoproteins in atherosclerotic lesions - Localization by immunofluorescence of ape-low density lipoproteins in human atherosclerotic arteries from normal and hyperlipoproteinemics. Arch. Path., 99 (1975) 253. Hoff, H.F.. Heideman. C.L., Gaubatz, J.W., Scott. D.. Titus, J.L. and Gotto, A.M., Correlation of apolipoprotein B retention with the structure of atherosclerotic plagues from human aortas, Lab. Invest., 38 (1978) 560. Smith, E.B. and Slater, R.S., The chemical and immunological assay of low-density lipoproteins extracted from human aortic intima, Atherosclerosis, 11 (1970) 417. Smith, E.B. and Smith, R.H., Early changes in aortic intima. Atheroscler. Rev., 1 (1976) 119. Smith, E.B., Ma&e. I.B. and Alexander, K.M., The release of an immobilized lipoprotein fraction from atherosclerotic lesions by incubation with plasmin. Atherosclerosis, 25 (1976) 71. Restrepo, C. and Tracy, R.E., Variations in human aortic fatty streaks among geographic locations, Atherosclerosis, 21 (1975) 179. Hollander, W., Unified concept of the role of acid mucopolysaccharides and connective tissue proteins in the accumulation of lipids, lipoproteins, and calcium in the atherosclerotic plaque, EXP. Mol. Path., 25 (1976) 106. Hoff, H.F., Karagas, M., Heideman, CL., Gaubatz, J.W. and Gotto, Jr.. A.M., Correlation of looselyand tightly-bound apo B in the human aorta with tissue cholesterol and collagen content, Submitted.
Atherosclerosis, 30 (1978) 263-272 Scientific @ Elsevier/North-Holland
QUANTITATION
Publishers,
Ltd.
OF APO B IN HUMAN AORTIC FATTY STREAKS
A Comparison with Grossly Normal Intima and Fibrous Plaques HENRY F. HOFF, CAROL L. HEIDEMAN, ANTONIO M. GOTTO, JR. Departments (U.S.A.)
JOHN W. GAUBATZ,
JACK L. TITUS and
of Medicine and Pathology, Baylor College of Medicine, Houston, TX 77030
(Received 12 December, 1977) (Revised, received 20 March, 1978) (Accepted 19 April, 1978)
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 (E.cgper 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 f 0.15. Using a paired t-test, buffer-extracted apo B was marginally significantly greater in the adjacent normal intima than fatty streaks (P < 0.05), whereas Triton-extracted apo B was highly significantly greater in fatty streaks than adjacent normal intima (P < 0.0005). When the mean apo B values of these 18 fatty streaks and 23 fibrous plaques from separate cases were compared in a non-paired t-test, buffer-extracted apo B was slightly higher in fatty streaks than fibrous plaques (P < 0.025), whereas Triton-extracted apo B was much higher in fibrous plaques than in fatty streaks (P < 0.0005). The intermediate position of fatty streaks between grossly normal and fibrous plaques with respect to buffer- and Triton-extracted apo B content, gives additional support to the contention that this lesion is an intermediate step in the progression of the grossly normal intima to a fibrous plaque. Assuming this sequence of progression to occur, our results demonstrate a marginally signifiSupported by the National Heart and Blood Vessel Research and Demonstration Center, Baylor College of Medicine (NIH Grant HL-17269). NIH Grant NS-09287 and a Grant in Aid from the American Heart Association. Dr. Hoff is an Established Investigator of the American Heart Association. Send reprint requests to: Dr. Henry F. Hoff. Department of Medicine, Baylor College of Medicine, Houston, TX 77030. U.S.A.
264
cant decrease in buffer-extracted apo B but a highly statistically increase in Triton-extracted apo B with lesion development. Key words:
Aortas - Apolipoprotein B (APO B) - Atherosclerosis -Low density lipoproteins -Man
significant
- Electroimmunoassay
Introduction In the progression of atherosclerosis in man the fatty streak lesion has long been considered to be the precursor of the fibrous plaque [ 11 as defined by WHO criteria [ 21. However, in recent years this point has become increasingly controversial [ 1,3]. Supportive of this contention are data from experimental animal models in which atherosclerosis is induced by feeding diets high in cholesterol and saturated fats [4,5]. Early lesions in animals resemble the human fatty streaks and later appear as a more advanced lesion usually resembling human fibrous plaques [ 1,4,5]. Changes intermediate between the two lesions have been documented in human arteries by conventional morphology [6], enzyme histochemistry [ 71, and physical chemistry of the lipids in the lesions [8]. On the other hand, while both lesions are located at the same anatomic sites of coronary arteries in man [ 1,3,9], fibrous plaques of the aorta in a later decade of life usually do not develop at the same geographic locations where fatty streaks are found during the second and third decades of life [lo]. Moreover, the major fatty acid in the cholesterol ester fraction of fatty streaks is oleic acid whereas in fibrous plaques it is linoleic acid [ 11,121, as it is in plasma low density lipoproteins (LDL) [ 131. In a recent study, we quantitatively measured apolipoprotein B (apo B) the major protein of LDL, in both the grossly normal aortic intima [14] and in aortic fibrous plaques [ 151. Most of the accumulated apo B in the normal intima is extractable from a tissue homogenate with a standard buffer or saline [ 141. However, in aortic plaques only half of the apo B is extractable with a conventional buffer, the remainder extractable with the detergent Triton X-100 [16]. Conceivably, if fatty streaks are precursors of the fibrous plaque, then the amount of apo B present in buffer- or Triton-extractable forms would be intermediate between grossly normal intima and aortic plaques. In this report we have documented the amounts of buffer- and Triton-extracted apo B in 18 human aortic fatty streaks and demonstrated that these values lie intermediate between those found in adjacent normal intima and those found in 23 fibrous plaques from separate cases. Material and methods Each aortic sample was obtained at autopsy from the County Medical Examiner within 18 h after death. Most cases were trauma victims. Fatty streak lesions and adjacent grossly normal regions were excised from the descending thoracic aorta from 18 males between the ages of 21 and 67 years. Fibrous plaques [2] were also removed from the aorta of 23 males between the ages of 39 and 72 years. Grossly normal regions located between the plaques were
265
present in 10 of the 23 cases and were also removed. The size of the intimal surface of grossly normal intima removed ranged from 10 to 30 cm2 depending on the thickness of the intima. The intimal lining from each tissue specimen was stripped from the underlying tunica media, finely minced, and homogenized in a standard Tris-EDTA buffer pH 7.4 using a Polytron homogenizer (Brinkmann, New York) as described in detail earlier [ 14,151. Aliquots of the supernatant fraction of this homogenate were measured for buffer-extracted apo B content using an electroimmunoassay (EIA) [ 14,171. The pellets of such homogenates extracted twice with buffer were re-extracted with 3% Triton X-100 in the standard Tris buffer to remove any residual apo B which could not be extracted with a standard buffer. Aliquots of this supernatant fraction were applied to an EIA using LDL treated with Triton as a standard. The details of the procedure to quantitate the Triton-extracted apo B fraction have been reported elsewhere [ 161. Representative regions of each sample of grossly normal intima, fatty streak, or fibrous plaque were taken both for conventional microscopy, i.e. Movatstained [18] sections of paraffin-embedded specimens, and for cryostat sections which were stained for lipid with oil red 0 and apo B with an immunofluorescence procedure [ 19,201. A Leitz Orthoplan photomicroscope was employed in conjunction with a Leitz Ploem illumination system for fluorescence microscopy and interference contrast system for bright field microscopy. Results
General morphologic description The smooth-surfaced intimal lining of the grossly normal human aorta from adults demonstrated thickness varying from approximately 50-150 I.tm. As shown previously the intima was characterized by the presence of smooth muscle cells in a matrix of collagen fibers and proteoglycans with reduplicated or fragmented elastica present at the intima-media border [ 141. Lipid was also localized in this area usually associated with connective tissue fibers. Apo B was often present diffusely localized in the thickened intima of adults, but not in the tunica media [14]. Fatty streak lesions from adults were characterized grossly as small, slightly raised yellow regions, surrounded by the smoother and whiter grossly normal intima (Fig. la). Microscopically this lesion demonstrated intimal thickening, slightly greater than corresponding grossly normal areas (90-200 pm). Numerous foam cells with both round and spindle shapes were present in the intima, as well as monocytes (Fig. lb, c), collagen fibers, proteoglycans and some reduplicated or fragmented elastic fibers (Fig. Id). Oil red O-stainable lipid was present mainly within foam cells, but also extracellularly (Fig. le), whereas apo B was present diffusely in the extracellular space (Fig. If). Pellets of homogenates of either fatty streaks or normal intima were negative for apo B by immunofluorescence following two extractions with Triton. As demonstrated in earlier studies the grossly raised fibrous plaque usually had a necrotic core containing lipid gruel demonstrable by oil red 0 stain at its base and a fibrotic or fibromuscular cap on the lumen surface [2,15]. The topographical distribution of apo B, as seen in earlier studies [19,20] was
266
Fig. 1. (I: gross morphology of fatty streaks in human thoracic aorta from a 23-year-old individual. Note the rows of small raised areas. X1.2. b: micrograph of a Movat-stained section of a fatty streak lesion using interference contrast optics. Note the presence in the intima of numerous foam cells and blood monocytes. L = lumen. X120. c: higher magnification of fatty streak depicting the vacuolated appearance of the foam cells close to the lumen border. Interference contrast optics. L = lumen. X 300. d: micrograph of the intima-media border of a fatty streak lesion depicting the reduplicated elastica (black lines) on the intimal side together with numerous collagen fibers seen as bundles of wavy material. Some of the bundles stained positively with alcian blue suggesting the association of glycossminoglycans with some of the collagen. X300. e: oil red O-stained cryostat section of a fatty streak. The dark droplets depicting lipid can be seen as large spheres close to nuclei. presumably representing intracellular lipid, as well as extracellularly localized smaller spheres (arrow). X180. f: localization of apo B (white) only in the intima of this fatty streak. Elastic fibers in media autofluorescent. Immunofluorescence stain. L = lumen. X 100.
found often together with oil red O-stainable lipids as a diffuse array in the necrotic core and along connective tissue fibers in the fibromuscular cap. For quantitative studies we utilized only one plaque from each case, and only lesions which by microscopy demonstrated lipid-filled necrotic cores which encompassed over 50% of the total cross-sectional area [ 211.
267
Quan titation of apo B The buffer- and Triton-extracted apo B content in c(g per mg tissue dry weight of fatty streaks from the thoracic aortas of 18 individuals was compared to values obtained from the adjacent grossly normal intima (Table 1). By statistical analysis (paired t-test) the buffer-extracted apo B content in the grossly normal intima (mean value: 6.51 f 0.92) was only marginally significantly greater (P < 0.05) than the corresponding value in fatty streaks (mean value: 4.67 + 0.51). In cast 5 cases (2, 3, 4, 8 and 15) showed the opposite trend. TABLE 1 BUFFERGROSSLY Case NO.
AND TRITON-EXTRACTED NORMAL INTIMA Age/ Race
Sample a
2
21w 21w
3
21b
4
22w
5
23b
6
26~
7
27~
8
29w
9
29w
10
33w
11
33b
12
37b
13
39w
14
41w
15
44b
16
52~
17
57w
18
67~
Mean f SEM
34 + 3
--
N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS N FS
FATTY
STREAKS
AND
ADJACENT -
Triton-extracted apo B
Buffer-extracted ape B Wmg
1
APO B IN AORTIC
D.W.
5.65 2.01 0.92 4.07 0.55 2.50 0.75 2.81 7.72 7.05 9.23 7.47 6.56 4.73 2.78 9.36 7.00 3.97 11.98 7.41 5.90 4.66 3.63 1.40 9.92 2.82 11.38 5.19 2.48 3.87 10.83 3.37 10.82 6.26 9.26 5.19 6.51 + 0.92 4.67 f 0.51 e P < 0.05
9%of total
Wmg
100 100 100 100 57 100 45 90 74 83 78 84 61 70 65 100 a4 100 96 100 88 100 100 100 54 90 50 100 61 100 81 100 95 100 62 94.6 75.1
0 0 0 0 0 1.91 0 3.42 0.90 2.41 1.94 2.01 1.20 3.00 1.16 5.03 0 0.76 0 0.21 0 0.65 0 0 0 2.40 1.41 5.20 0 2.43 0 0.81 0 0.30 0 3.14 0.37 f 0.15 1.88 f 0.39 o P < 0.0005
.
D.W.
% of total 0 0 0
0 0 43 0 55 10 26 17 22 16 39 30 35 0 16 0 4 0 12 0 0 0 46 10 50 0 39 0 19 0 5 0 38 5.4 24.9
a N = grossly normal intima; FS = fatty streak. b In a paired f-test differences between normal and fatty streak group were statistically significant at the levels indicated.
268
However, in a paired t-test the Triton-extracted apo B content of fatty streaks (mean value: 1.88 f 0.39) was highly significantly greater (P < 0.0005) than that of adjacent normal areas (mean value: 0.37 + 0.15). The grossly normal intima of 13 of the 18 cases did not contain measurable amounts of Tritonextracted apo B. Numerous fatty streaks contained Triton-extracted apo B values over 3 (ug per mg tissue dry weight (cases 4, 7, 8, 14 and 18). In some of these lesions patchy areas of necrosis were seen by microscopy. Extracellular lipid and apo B distribution was also more widespread in fatty streaks from older individuals than younger ones. Although the width of each thickened intima increased with age in the grossly normal intima, no positive correlation could be found between either buffer- or Triton-extracted apo B per unit dry weight and intimal thickening in either normal intima or fatty streaks. We also compared the buffer- and Triton-extracted apo B content of the fatty streaks with that of fibrous plaques from a separate group of cases (Table 2). The mean buffer-extracted apo B content of the 18 fatty streaks (4.67 + 0.51) was higher than that of 23 fibrous plaques (3.18 + 0.41), but using a nonpaired t-test the level of statistical significance was marginal (P < 0.025). By contrast the mean Triton-extracted apo B content of the fibrous plaques (3.78 + 0.31) was highly significantly greater than that of fatty streaks (1.88 + 0.39). The level of significance was P < 0.0005. Finally in a series of grossly normal intimas comprising both the 18 normal regions adjacent to the fatty streaks plus 10 additional cases, we compared the buffer- and Triton-extracted apo B content from cases 39 years of age or above (>39) with those below 39 years of age (<39). This age was chosen because cases 39 years or over demonstrated the presence of raised aortic fibrous plaques, whereas the cases below 39 years of age did not demonstrate the presence of such lesions. As seen in Table 3, the normal intima of the group >39 years had mean buffer-extracted apo B values (7.66 f 0.84) marginally siginificantly greater (P < 0.05) than that of normal intima of the group <39 years (5.12 f 0.96). No significant differences were detected for Triton-extracted apo B values between the two groups. Comparing normal and atherosclerotic intima for the amount of apo B in loosely- and tightly-bound fractions (Tables 1 and 2) permitted the following statistically significant observations: for buffer-extracted apo B fractions, grossly normal intima (239 years) > grossly normal intima (<39 years) > fatty
TABLE 2 COMPARISON OF THE MEAN BUFFER- AND MEAN FATTY STREAKS AND FIBROUS PLAQUES Lesion
Fatty streak Fibrous plaque
Mean age f SEM
34 + 3 56 f 4
Number of cases
18 23
TRITON-EXTRACTED
APO B VALUES
Mean apo B value (pg per mg tissue dry weight) f SEM Buffer-extracted
T&on-extracted
4.67 f 0.51 a 3.18 + 0.41 a
1.88 * 0.39 b 3.78 + 0.31 b
a Statistically significant difference using non-paired t-test: P < 0.025. b Statistically significant difference using non-paired t-test: P < 0.0005.
IN
269 TABLE 3 COMPARISON OF THE MEAN BUFFER- AND MEAN TRITON-EXTRACTED APO B VALUES IN THE GROSSLY NORMAL AORTIC INTIMA FROM CASES BELOW AND ABOVE 39 YEARS OF AGE Mean age 2 SEM
Number of cases
Mean ape B value (pg per mg tissue dry weight) f SEM
Normal (>39 Normal (<39
yrs) yrs)
52 + 3 27 + 2
15 13
Buffer-extracted
Triton-extracted
7.66 i 0.84 a 5.12 i- 0.96 a
0.16 i- 0.10 b 0.43 f 0.20 b
a Differences statistically significant: P < 0.05. b Differences not statistically significant.
streaks > fibrous plaques; for Triton-extracted apo B fractions, fibrous plaques > fatty streaks > normal intima. It should be noted that only for the Tritonextracted apo B groups were the differences highly statistically significant (P < 0.0005). Discussion We have compared the apo B content of human fatty streaks from the thoracic aorta of 18 males with that of adjacent grossly normal intima as well as with that of fibrous plaques from 23 separate cases. Smith et al. [22,23] had previously compared values of apo B (expressed as LDL cholesterol) in human aortic fatty streaks with those in adjacent grossly normal intima. They also found lower values for apo B in fatty streaks than in adjacent normal intima, as we have shown, but the differences were much greater than in our study. The discrepancy in results could be due to the differences in techniques employed i.e. Smith et al. [22,23] inserted minces of aortic intima directly into wells of EIA, whereas we pipeted ahquots of supernatant fractions of tissue homogenates, a procedure we have shown to measure more apo B than when applying minces directly to the assay. The discrepancy could also be due to the preferential dissection by Smith et al. of focal regions of fatty streaks containing large accumulations of foam cells essentially free of extracellular material. By contrast the tissue used by us in this study contained both regions filled with foam cells as well as large regions of extracellular space. Recently Smith et al. [24] demonstrated that a substantial amount of residual apo B could be measured from minces of raised atherosclerotic lesions which had previously been applied to an EIA. This was accomplished by treating aortic minces with such hydrolytic enzymes such as plasmin or collagenase and then re-assaying. In some plaques the residual apo B was about the same amount as the buffer-extracted fraction of apo B [ 241. It is quite possible that residual apo B was also present in the minces of fatty streaks, which would correspond to the Triton-extracted apo B fraction in our study. In a previous report we have presented evidence demonstrating that the combined treatment of arterial homogenates with a conventional buffer, followed by one containing Triton, quantitatively removed all apo B from the arterial tissue [ 161. In those studies we had shown that grossly raised fibrous
270
plaques contain roughly equal amounts of buffer- and Triton-extracted apo B, whereas adjacent grossly normal intima contained primarily buffe;-extracted apo B. In this present study, fatty streak lesions contained significantly higher values of residual apo B, extractable with Triton X-100, than adjacent normal areas. Fatty streaks were intermediate between grossly normal intima and fibrous plaques with respect to both buffer- and Triton-extracted apo B. These findings suggest that the fatty streak may be an intermediate step in the progression from a grossly normal intima to a fibrous plaque [ 1,3]. Differences in topographical distribution of fatty streaks earlier and fibrous plaques later in life on the aortic lumen surface [ 1,3,10] do not necessarily preclude a precursor-product relationship between the two. They merely suggest that some fatty streaks may develop into fibrous plaques while others regress. Significant correlations, however, were found between the appearance of aortic fatty streaks in the second decade of life containing small amounts of necrosis or monocytes and the appearance of fibrous plaques in the same location after the fourth decade [ 251. Differences in the fatty acid pattern of cholesterol esters in the fatty streaks and fibrous plaques [11,12] has been used as evidence refuting a precursorproduct relationship between these two lesions [ 1,3]. However, greater retention of the linoleic acid-rich plasma LDL by the fatty streak as it progresses might have diluted out the oleic acid-rich content of the original fatty streak which contains predominantly intracellular lipid. Katz et al. [S] have demonstrated the occurrence of aortic lesions in man in which the physical-chemical state of the lipid was intermediate between that found in fatty streaks and fibrous plaques. Such lesions contained both foam cells and extra-cellular pools of lipid. The quantitative results of apo B retention in this study suggest that, as an atherosclerotic lesion develops, so does the percent of total apo B which must be extracted with Triton. This is consistent with the results of an earlier study from this laboratory showing that the lipid core at the base of advanced lesions was the major region of Triton-extracted apo B in such plaques [ 221. When we compare the values in this present study with those obtained in the earlier study on plaque lipid cores and fibrotic capsules separated by microdissection, we find that lipid cores have the highest content of Triton-extracted apo B per unit dry weight. Also, even though most apo B in the fibrotic capsule was loosely-bound, the amount of Triton-extracted apo B in these capsules was greater than that in fatty streaks even though the difference was only significant at the P < 0.05 level. The finding that the Triton-extracted fraction of apo B in fatty streaks was larger than that of grossly normal intima from cases over 39 years of age suggests that the retention of this fraction of apo B is diseaserather than age-related. Little is yet known about the characteristics of the Triton-extracted fraction of apo B. It seems unlikely that the material is insoluble in aqueous buffer due to delipidation of apo B, since extracellular lipid is plentiful in both fatty streaks and grossly normal intima. Moreover, we have been able to liberate aqueous soluble apo B from pellets of plaque homogenates by digesting with elastase (unpublished observations) instead of treatment with Triton. Similar approaches were taken by Smith et al. [24] and Hollander [25]. These results
271
suggest that a portion of the Triton-extracted apo B was at least partially lipidated. Further preliminary data [27] has demonstrated significant positive correlations between this fraction of apo B and tissue cholesterol. Similar results were obtained by Smith et al. [24]. These data suggest the possibility that cholesterol is involved in the tight-binding of certain of the apo B-containing lipoproteins which insudate into the arterial wall. Further studies are required before the mode of apo B retention in the arterial wall is completely elucidated. Acknowledgments The authors wish to acknowledge the help of members of the Harris County Medical Examiner’s Office in aiding in the procurement of the aortic samples, and Dr. Henry C. McGill, Professor of Pathology, University of Texas Health Science Center, San Antonio, for constructive criticism in the preparation of the final manuscript. References 1 McGill, Jr., H.C., The lesion in atherosclerosis. In: G. Schettler and A. Weizel (Eds.), II Proc. of the Third International Symposium, Springer-Verlag, New York, 1974, pp. 27-38. 2 Classification of Atherosclerotic Lesions. Report of a study group: WHO Tech. Rep. Ser., 143 (1958) 3. 3 McGill, Jr., H.C., Atherosclerosis -Problems in pathogenesis, Atheroscler. Rev., 2 (1977) 27. 4 Clarkson, T.B. and Lofland, H.B., Effects of cholesterol-fed diets on pigeons susceptible and resistant to atherosclerosis, Circ. Res., 9 (1961) 106. 5 Scott, R.F., Daoud. AS. and Florentin. R.A.. Animal models in atherosclerosis. In: R.W. Wissler and J.C.Geer (Eds.), The Pathogenesis of Atherosclerosis. Williams and Wilkins, Baltimore, MD, 1972. pp. 120-146. 6 Geer, J.C.. McGill, Jr., H.C., Robertson. W.B. and Strong, J.P., Histologic characterization of coronary artery fatty streaks, Lab. Invest.. 18 (1968) 565. 7 Hoff, H.F., A histoenzymatic study of human intracranial atherosclerosis, Amer. J. Path., 67 (1972) 583. 8 Katz. S.S., Shipley, G.G. and lesions - Demonstration of a Invest., 58 (1976) 200. 9 Montinegro, M.R. and Eggan, 18 (1968) 586. 10 Mitchell, J.R.A. and Schwartz, 11 12 13
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